Inhibition of bispecific monoclonal antibody (bsAb)-targeted cytolysis by human anti-mouse antibodies in ovarian carcinoma patients treated with bsAb-targeted activated T-lymphocytes

T lymphocytes of 8 patients with ovarian cancer were targeted to the tumor cells using F(ab')₂ fragments of a bispecific monoclonal antibody (bsAb), specific for CD3 (a component of the T lymphocyte receptor for antigen) and for the folate receptor MOv 18 (overexpressed by ovarian carcinoma cells) as part of a phase l/II study. Phase I (days 0 to 3) consisted of increasing intraperitoneal (i.p.) numbers (10⁶?10⁹) of bsAbtargeted T lymphocytes plus lowdose interleukin-2 (IL-2). Phase II (days 6 to 13, and 27 to 33) consisted of daily i.p. infusions of 10⁹ targeted T lymphocytes, 2 mg soluble bsAb, and lowdose IL-2. Using enzymelinked immunosorbent assays (ELISA), human antimouse antibodies (HAMA) were detected in all patients: in the serum from day 13 onwards and in the peritoneal fluid from day 20 onwards. A significant proportion of the HAMA appeared to be directed against the idiotypes of the bsAb specific for CD3 and MOv18, as suggested by (I) the clearly higher ELISA titers against OC/TR bsAb as compared to those against a monoclonal antibody (MAb) with unrelated specificity, and (2) failure to abrogate the capacity of peritoneal fluid containing HAMA to block the binding of OC/TR bsAb to MOv18⁺ or CD3⁺ cells by absorption of human antimouse IgG-framework antibodies in peritoneal fluid to immobilized mouse IgG. The OC/TR-targeted cytolysis of the MOv18⁺ ovarian carcinoma cell line lgrov-1 by autologous T lymphocytes was inhibited by peritoneal fluid samples containing relatively high HAMA titers. Such inhibitory activity was never detected at the start of phase II, but coincided with the last series of i.p. infusions of targeted T lymphocytes in 2 patients.     

Pharmacokinetics,biodistribution and biological effects of intravenously administered bispecific monoclonal antibody OC/TR F(ab′)2 in ovarian carcinoma patients

The bispecific monoclonal antibody (biMAb) OC/TR combines the anti-ovarian-cancer reactivity of the MOv18 monoclonal antibody (MAb) with the reactivity of an anti-CD3 MAb. Pre-clinical studies have indicated that this biMAb is able to redirect the cytolytic activity of T cells towards tumour cells, resulting in efficient tumour-cell lysis. To assess the clinical potential of systemic biMAb-based cancer therapy we initiated a study in ovarian-cancer patients. Five patients suspected of ovarian cancer received ¹²³I-OC/TR F(ab′)₂ i.v. Unexpectedly, the first patient developed side effects (grade III–IV toxicity) starting 30 min after infusion (p.i.) of 1 mg of OC/TR F(ab′)₂. After approval of the Ethical Committee, the study was continued at lower dose levels (0.1 mg; 0.2 mg). However, at the 0.2-mg dose level similar side effects were observed. FACS analysis indicated that all peripheral T cells were coated with biMAb immediately following the infusion. The cytokines tumour necrosis factor-α, interferon-γ and interleukin-2 showed maximum serum concentrations 2 h p.i. Tumour uptake ranged from 0.8 to 1.9% ID/kg, resulting in tumour/background ratios of 3 to 8. Our results suggest that at higher antibody dose levels OC/TR F(ab′)₂ causes T-cell activation with acute release of cytokines. Only low doses of biMAb can be administered safely. Despite the interaction with T cells, OC/TR F(ab′)₂ preferentially localizes in tumours following i.v. administration, thus offering therapeutic perspectives. © 1996 Wiley-Liss, Inc.     

Analysis of production,purification, and cytolytic potential of bi-specific antibodies reactive with ovarian-carcinoma-associated antigens and the T-cell antigen CD3

OV-TL 3 and MOvl8 MAbs, due to their restricted specificity, have been successfully used to visualize ovarian cancer in patients and might therefore be used to develop therapies for ovarian cancer. The bi-specific MAbs αT3/OC2 and αOC/TR (both being combinations of MOvl8 and aCD3) have been shown to lyse ovarian tumor cells in vitro. To evaluate the relative merits of MOvl8/CD3 and OV-TL 3/CD3, the present study was undertaken in which the bi-specific MAbs αT3/OC2 and αOC/TR, and a newly developed bi-specific MAb, OV-TL 3/CD3, were highly purified and compared for specificity, stability, purification and cytolytic potential. The dual specificity of the hybrid-hybridoma supernatants was analyzed by immunohistochemistry, and by testing bi-specific MAb-mediated cytotoxicity against relevant target cells in the presence of effector cells. Stability testing of bi-specific MAb-producing hybrid-hybridomas showed that, after sub-cloning, clones stably produced up to 40% bi-specific MAb even after prolonged in vitro culture. The purification of the bi-specific fractions was performed with protein A and by ion-exchange high-pressure liquid chromatography, depending on the sub-class combination of the bi-specific MAb. The purified bi-specific MAbs were tested for their ability to mediate target-cell lysis with the use of cytotoxic T-cell clones and activated peripheral-blood lymphocytes. The purified αT3/OC2, αOC/TR, and OV-TL 3/CD3 were all able to mediate highly specific lysis of various ovarian-carcinoma cell lines. No correlation was found between the level of antigen expression and bi-specific MAb-mediated cytolysis. © 1993 Wiley-Liss, Inc.     

Discovery of chemotherapy‐associated ovarian cancer antigens by interrogating memory T cells

According to the immunogenic cell death hypothesis, clinical chemotherapy treatments may result in CD8⁺ and CD4⁺ T‐cell responses against tumor cells. To discover chemotherapy‐associated antigens (CAAs), T cells derived from ovarian cancer (OC) patients (who had been treated with appropriate chemotherapy protocols) were interrogated with proteins isolated from primary OC cells. We screened for immunogenicity using two‐dimensional electrophoresis gel‐eluted OC proteins. Only the selected immunogenic antigens were molecularly characterized by mass‐spectrometry‐based analysis. Memory T cells that recognized antigens associated with apoptotic (but not live) OC cells were correlated with prolonged survival in response to chemotherapy, supporting the model of chemotherapy‐induced apoptosis as an adjuvant of anti‐tumor immunity. The strength of both memory CD4⁺ and CD8⁺ T cells producing either IFN‐γ or IL‐17 in response to apoptotic OC antigens was also significantly greater in Responders to chemotherapy than in nonresponders. Immunogenicity of some of these antigens was confirmed using recombinant proteins in an independent set of patients. The T‐cell interrogation system represents a strategy of reverse tumor immunology that proposes to identify CAAs, which may then be validated as possible prognostic tumor biomarkers or cancer vaccines.     

Local but no systemic immunomodulation by intraperitoneal treatment of advanced ovarian cancer with autologous T lymphocytes re-targeted by a bi-specific monoclonal antibody

We have reported a 27% overall anti-tumor response using i.p. immunotherapy of advanced ovarian carcinoma with autologous, ex vivo expanded, T lymphocytes re-targeted with bi-specific monoclonal antibody OC/TR, combined with soluble OC/TR and low-dose recombinant interleukin-2 (IL-2). This treatment had no effect on extraperitoneal disease. Therefore we studied in 13 patients whether this immunotherapeutic protocol resulted only in local or also in systemic immunomodulation. The phenotype of the ex vivo expanded lymphocytes was mainly CD3⁺, 4⁻, 8⁺, 16⁻, 56⁻. Their OC/TR-re-targeted cytolytic activity against Igrov-1 ovarian-carcinoma cells was approximately as high in responders as in non-responders. Following most therapeutic cycles, the immunophenotype of lymphocytes recovered from the peritoneal fluid was similar to that of the infused T cells (i.e., mainly CD3⁺, 4⁻, 8⁺) and they were coated with OC/TR. However, cytolytic activity of the recovered lymphocytes against Igrov-1 cells was low in direct assays, and only slightly increased after additional in vitro re-targeting with OC/TR. Systemically, the i.p. immunotherapy resulted in a transient lymphopenia lasting for about 7 days, low (i.e., 5 to 13 ng/ml) serum concentrations of free, functional OC/TR, and very weak coating of circulating T lymphocytes with OC/TR. These peripheral-blood T lymphocytes did not exert OC/TR-re-targeted cytolytic activity. Thus, locoregional OC/TR-re-targeted cellular immunotherapy resulted in substantial local immunomodulation and anti-tumor effects but virtually no systemic immunomodulation. Int. J. Cancer 73:211–219, 1997. © 1997 Wiley-Liss, Inc.     

Generation of chimeric bispecific G250/anti-CD3 monoclonal antibody, a tool to combat renal cell carcinoma.

The monoclonal antibody (MAb) G250 binds to a tumour-associated antigen, expressed in renal cell carcinoma (RCC), which has been demonstrated to be a suitable target for antibody-mediated immunotherapy. A bispecific antibody having both G250 and anti-CD3 specificity can cross-link G250 antigen-expressing RCC target cells with T cells and can mediate lysis of such targets. Therapy studies with murine antibodies are limited by immune responses to the antibodies injected (HAMA response), which can be decreased by using chimeric antibodies. We generated a chimeric bispecific G250/anti CD3 MAb by transfecting chimeric genes of heavy and light chains for both the G250 MAb and the anti-CD3 MAb into a myeloma cell line. Cytotoxicity assays revealed that the chimeric bispecific MAb was capable of mediating lysis of RCC cell lines by cloned human CD8+T cells or by IL-2-stimulated peripheral blood lymphocytes (PBLs). Lysis mediated by the MAb was specific for target cells that expressed the G250 antigen and was effective at concentrations as low as 0.01 microgram ml-1. The chimeric bispecific G250/anti-CD3 MAb produced may be an effective adjuvant to the currently used IL-2-based therapy of advanced renal cell arcinoma.     

Anti‐VEGF antibody enhances the antitumor effect of CD40

As its central immunomodulatory effects, CD40 induces interleukin (IL)‐12‐dependent antitumor immune responses; as its local protumor effects, CD40 induces the expression of vascular endothelial growth factor (VEGF) that promotes tumor angiogenesis and growth. Therefore, using a previously established tumor model in mouse, we examined if the antitumor functions of CD40 are self‐limited by VEGF induction. We observed that as the tumor mass grew during day 6 to day 18, VEGF expression in the tumor peaked with concomitant decrease in expressions of CD40 and IL‐12 but not of IL‐10. Among the angiogenic factors, VEGF‐B, VEGFR‐1, VEGFR‐2, angiopoietin‐1 and Tie2 expressions decreased, whereas the expressions of angiopoietin‐2 and angiopoietin‐3 increased with tumor growth. As significant changes in the expressions of these factors were observed on day 6, we treated the tumor‐bearing mice with the agonistic anti‐CD40 antibody or neutralizing anti‐VEGF antibody—alone or in combination—from the fifth day after the injection of tumor cells. The anti‐VEGF antibody significantly enhanced the antitumor effects of the anti‐CD40 antibody, as observed through increased survival of the mice, accompanied by reduced angiogenesis and angiopoietin‐2 expression but higher T‐cell proliferation in response to tumor antigens, increased interferon‐γ production and tumor cell cytotoxicity and higher levels of tumor antigen‐specific serum IgM, IgG1 and IgG2a, indicating B‐cell activation. Thus, our data show for the first time that the combined treatment with an agonistic anti‐CD40 antibody and a neutralizing anti‐VEGF antibody, which increases antitumor immune response or reduces local angiogenesis, respectively, is a novel antitumor strategy.     

A radioimmunoassay for the detection of a human tumor-associated glycoprotein (TAG-72) using monoclonal antibody B72.3

Monoclonal antibody (MAb) B72.3 was generated against a carcinoma metastasis and has been shown to bind with a high degree of selectivity to a tumor-associated glycoprotein (TAG-72) found in human colon and breast carcinomas, while showing minimal reactivity to any normal adult tissues. Competition radioimmunoassays have been developed for the detection of TAG-72 in tumors and sera from both athymic mice bearing human carcinoma xenografts and patients with colon, breast and other carcinomas. The distribution of TAG-72 in human tumor xenografts was restricted to tumors originating from the LS-174T human colon carcinoma, with no significant reactivity being detected in human tumor xenografts from a different colon carcinoma, a human breast carcinoma, or a human melanoma. The levels of TAG-72 in clinical material obtained from surgery were examined; high levels were found in colon carcinomas and to a lesser extent in breast carcinomas, while no detectable levels were found in normal tissues. Sera from apparently normal patients contained a mean level of 2.2 units per ml of TAG-72. A level of 3 standard deviations above the mean level of TAG-72 found in normals was employed as a cut-off value to indicate a positive test result in subsequent studies. No patient with inflammatory disease or other benign colon disease exhibited elevated levels of TAG-72. Seven out of 20 patients with other carcinomas had elevated serum levels of TAG-72. The serum TAG-72 levels were compared with the serum levels of antigens recognized by the MAbs currently used to screen sera of carcinoma patients (CEA, GICA, and OC125). It was clearly demonstrated that TAG-72 is different from these antigens and can be found in some sera in which no antigen is detected by otherwise available MAb RIAs.     

卵巢癌抗独特型微抗体疫苗诱导BALB/c小鼠体内抗肿瘤免疫应答的实验研究

背景与目的：6B11抗独特型微抗体由模拟人卵巢癌抗原的抗独特型单链抗体(6B11ScFv)融合人IgG1铰链区和CH3区所构成,它具有6811ScFv和人1gGFc的双重免疫学活性。本研究观察6811抗独特型微抗体在BALB／c小鼠体内诱导抗肿瘤免疫反应情况,探讨其作为卵巢癌疫苗的可能性。方法：用卵巢癌6B11抗独特型微抗体免疫BALB／c小鼠。采用间接ELISA、竞争抑制实验和流式细胞术检测免疫鼠血清。结果：6B11抗独特型微抗体免疫小鼠后,在不用佐剂的情况下可诱导小鼠产生较高的Ab3,末次免疫后30天仍持续在较高的水平。分别在末次免疫后4天、14天、24天和30天可刺激小鼠脾脏淋巴细胞CD4^ T细胞和CD8^ T细胞明显升高。结论：6811抗独特型微抗体可诱导机体产生特异体液免疫和细胞免疫反应,这为抗独特型微抗体疫苗的临床应用提供了一定的实验依据。     

Intermittent chemotherapy can retain the therapeutic potential of anti‐CD137 antibody during the late tumor‐bearing state

Immunomodulating monoclonal antibodies (mAb) can evoke antitumor T‐cell responses, which are attenuated by regulatory T cells (Treg) and myeloid‐derived suppressor cells (MDSC). Treatment with cyclophosphamide (CP) and gemcitabine (GEM) can mitigate the immunosuppression by Treg and MDSC, respectively. In the current study, we examined the antitumor effects of a combination of local injection with anti‐CD137 mAb and intermittent low‐dose chemotherapy using CP and GEM in subcutaneously established CT26 colon carcinoma. Although a significant antitumor effect was observed when local anti‐CD137 mAb therapy (5 μg) was started early in the tumor‐bearing stage (day 10), no therapeutic efficacy was observed when the mAb therapy was started at a later tumor‐bearing stage (day 17). Analyses of the tumor‐infiltrating immune cells revealed that the number of Gr‐1^high/low CD11b⁺ MDSC started to increase 13 days after tumor inoculation, whereas injection with low‐dose (50 mg/kg) CP and GEM mitigated this increase. In addition, although intermittent injections with low‐dose CP and GEM on days 10 and 18 suppressed tumor growth significantly, additional local injections of anti‐CD137 mAb on days 19, 21, and 23 further augmented the therapeutic efficacy. Cytotoxic T lymphocytes reactive to CT26 and a tumor antigen peptide were induced successfully from the spleen cells of tumor‐cured or tumor‐stable mice. In a bilateral tumor inoculation model, this combination therapy achieved systemic therapeutic effects and suppressed the growth of mAb‐untreated tumors. These results suggest that intermittent immunochemotherapy using CP and GEM could retain the therapeutic potential of anti‐CD137 mAb that is normally impaired during the late tumor‐bearing stage.     

Maintenance of long remission in adult T‐cell leukemia by Tax‐targeted vaccine: A hope for disease‐preventive therapy

Adult T‐cell leukemia/lymphoma (ATL) is an aggressive lymphoproliferative disease caused by human T‐cell leukemia virus type 1 (HTLV‐1). Multi‐agent chemotherapy can reduce ATL cells but frequently allows relapses within a short period of time. Allogeneic hematopoietic stem cell transplantation (allo‐HSCT) following chemotherapy is now a standard therapy for ATL in Japan as it can achieve long‐term remission in approximately one‐third of recipient ATL patients; however, it also has a risk of treatment‐related mortality. Allo‐HSCT often induces HTLV‐1 Tax‐specific cytotoxic T cells (CTL) as well as graft‐versus‐host (GVH) response in ATL patients. This observation led to development of a new therapeutic vaccine to activate Tax‐specific CTL, anticipating anti‐ATL effects without GVH response. The newly developed Tax‐DC vaccine consists of autologous dendritic cells pulsed with Tax peptides corresponding to CTL epitopes that have been identified in post‐allo‐HSCT ATL patients. In a pilot study of Tax‐DC therapy in three ATL patients after various initial therapies, two patients survived for more than 4 years after vaccination without severe adverse effects (UMIN000011423). The Tax‐DC vaccine is currently under phase I trial, showing a promising clinical outcome so far. These findings indicate the importance of patients’ own HTLV‐1‐specific T‐cell responses in maintaining remission and provide a new approach to anti‐ATL immunotherapy targeting Tax. Although Tax‐targeted vaccination is ineffective against Tax‐negative ATL cells, it can be a safe alternative maintenance therapy for Tax‐positive ATL and may be further applicable for treatment of indolent ATL or even prophylaxis of ATL development among HTLV‐1‐carriers.     

Activation of central/effector memory T cells and T‐helper 1 polarization in malignant melanoma patients treated with anti‐programmed death‐1 antibody

Human anti‐programmed death‐1 (PD‐1) antibody possesses the capability to revitalize host T cells and has been an effective therapy for metastatic malignant melanoma (MM). The precise subsets of T cells predominantly activated by anti‐PD‐1, however, have not yet been clarified. In this study, peripheral blood mononuclear cells obtained from MM patients scheduled to receive anti‐PD‐1 (nivolumab) therapy, and healthy subjects (HS), were systematically examined on flow cytometry to identify changes in the proportion of immune cell subsets. Compared with HS, MM patients prior to therapy had an increased proportion of activated CD8+ T cells with effector memory phenotypes (Tem), and PD‐1 positive subsets of CD4+ central memory T cells (Tcm) and T‐helper (Th)17 cells. After a single course of anti‐PD‐1 therapy, MM patients had an increase in activated Tem and Tcm subsets of CD4+ and CD8+ T cells, and activated Th1 plus T‐helper follicular 1 cells. There was no consistent change in the proportion of Tfh cells, B cells, natural killer cells, or dendritic cells. The observed activated phenotypes were attenuated during the course of therapy, but regulatory T cells belonging to the CD3+CD4+CD45RO+CD25high fraction increased at disease progression. Taken together, anti‐PD‐1 therapy modulates systemic immune reactions and exerts anti‐tumor effects, not only by revitalizing Tem and Tcm of CD4+ and CD8+ T cells, but also via a shift to a Th1 phenotype.     

NY‐ESO‐1 specific antibody and cellular responses in melanoma patients primed with NY‐ESO‐1 protein in ISCOMATRIX and boosted with recombinant NY‐ESO‐1 fowlpox virus

Vaccination strategies based on repeated injections of NY‐ESO‐1 protein formulated in ISCOMATRIX particles (NY‐ESO‐1 ISCOMATRIX) have shown to elicit combined NY‐ESO‐1 specific antibody and T cell responses. However, it remains unclear whether heterologous prime‐boost strategies based on the combination with NY‐ESO‐1 ISCOMATRIX with different NY‐ESO‐1 boosting reagents could be used to increase NY‐ESO‐1 CD8⁺ or CD4⁺ T cell responses. To address this question, we carried out a randomized clinical trial in 39 high‐risk, resected melanoma patients vaccinated with NY‐ESO‐1 ISCOMATRIX, and then boosted with repeated injections of either recombinant fowlpox virus encoding full length NY‐ESO‐1 (rF‐NY‐ESO‐1) (Arm A) or NY‐ESO‐1 ISCOMATRIX alone (Arm B). We have comprehensively analyzed NY‐ESO‐1 specific T cells and B cells response in all patients before and after vaccination for a total of seven time points per patient. NY‐ESO‐1 ISCOMATRIX alone elicited a strong NY‐ESO‐1 specific CD4⁺ T cell and antibody response, which was maintained by both regiments at similar levels. However, CD8⁺ T cell responses were significantly boosted in 3 out of 18 patients in Arm A after the first rF‐NY‐ESO‐1 injection and such responses were maintained until the end of the trial, while no patients in Arm B showed similar CD8⁺ T cell responses. In addition, our results clearly identified immunodominant regions in the NY‐ESO‐1 protein: NY‐ESO‐1_79–102 and NY‐ESO‐1_115–138 for CD4+ T cells and NY‐ESO‐1_85–108 for CD8+ T cells in a large proportion of vaccinated patients. These regions of NY‐ESO‐1 protein should be considered in future clinical trials as immunodominant epitopes.     

Patients treated with a monoclonal antibody (ab1) to the colorectal carcinoma antigen 17-1A develop a cellular response (DTH) to the "internal image of the antigen" (ab2)

The anti-tumor effector functions of unconjugated MAb in cancer therapy are not fully understood. Direct cytotoxic mechanisms have been demonstrated as well as induction of anti-idiotypic (ab2) and anti-anti-idiotypic (ab3) antibodies. If such a humoral response is induced, then an idiotypic cellular response would also be anticipated. Human monoclonal ab2s which mimic a tumor-associated antigen (TAA) (CO17-1A) of colorectal carcinoma (CRC) cells ("the internal image of the antigen") were produced. These ab2s were injected intradermally to patients with metastatic CRC who had been treated with the anti-colon carcinoma MAb 17-1A (ab1). Five out of 12 patients had a specific DTH (delayed-type hypersensitivity) reaction of the tuberculin type, which was proven by immunohistochemical analysis of skin biopsies. Serum ab3 was demonstrated in 4/4 tested DTH+ patients and also in 4 DTH patients. Control patients did not show any skin reactivity. Generation of an idiotypic response induced by the infused antibody (ab1) might be regarded as an active anti-tumor "vaccination". Induction of an idiotypic cellular and humoral cascade might be an important anti-tumor effector function of MAb and should be considered in future strategies for such therapy in cancer patients.     

Anti-murine Antibody Response to Mouse Monoclonal Antibodies in Cancer Patients

Although development of human anti-murine inununoglobulin antibody (HAMA) is often seen in patients receiving murine antibodies, the variety of methods used for detecting HAMA makes it difficult to compare directly the HAMA responses measured by different assays. In the present study, several parameters of the HAMA response to two murine monoclonal antibodies were evaluated. The anti-sialosyl Tn antibody MLS102 and anti-CA125 antibody 145-9, which were labeled with ¹¹¹ln, were injected intravenously into 17 colorectal cancer patients and 11 ovarian cancer patients for immnnoscintigraphy, respectively. HAMA was measured by enzyme-linked immunosorbent assay. There was no difference in baseline HAMA levels before antibody injection between the two groups. HAMA developed more frequently in ovarian cancer patients receiving the 145-9 antibody than in colorectal cancer patients receiving the MLS102 antibody (9/11 vs. 6/17, P <0.05). No significant difference was observed in maximal HAMA levels between the two groups of patients. However, time to reach the maximal levels was delayed and the duration of the response seemed longer in ovarian cancer patients. Among 11 patients receiving the 145-9 antibody three patients became positive for HAMA more than 2 months after antibody injection and the other two had HAMA activity in their sera for more than 17 months. HAMA response was different between the two antibodies, and late onset or long duration of HAMA response against the 145-9 antibody suggests the importance of HAMA measurement in patients who receive a second injection of murine antibodies even after a long interval.     

Antibody-antigen complex formation following injection of OC125 monoclonal antibody in patients with ovarian cancer

Monoclonal antibody (MAb) OC125 binds to approximately 80% of epithelial ovarian cancers. Serum antigen, CA125, can be detected in these patients. 131I-OC125-F(ab')2 was injected into 5 ovarian carcinoma patients with preinjection serum levels of 150 to 9,000 CA125 U/ml. Patients received the antibody intravenously in doses ranging from 0.46 to 0.94 mg with a specific activity of approximately 2.5 mCi/mg 131I. The half-life in the circulation was approximately 30 hr and was independent of serum CA125 levels. Clearance of 131I from the circulation fitted an open, one-compartment mathematical model. Gel filtration chromatography revealed antibody-antigen complexes in sera 15 min after injection of the radiolabelled antibody. By 5 days after injection, the free form of OC125 antibody could not be detected in the serum. The rate of complex formation correlated well with the observed preinjection serum CA125 levels. This direct correlation was verified in vitro using purified CA125 antigen and radiolabelled OC125 F(ab')2 fragments. The specific effects of complex formation on tumor localization remains unclear. However, the presence of complexes should not be ignored, when planning for diagnostic imaging or immunotherapy with OC125 or other MAbs reacting with circulating antigen.     

Role of α‐gal epitope/anti‐Gal antibody reaction in immunotherapy and its clinical application in pancreatic cancer

Pancreatic cancer is one of the most common causes of death from cancer. Despite the availability of various treatment modalities, such as surgery, chemotherapy and radiotherapy, the 5‐year survival remains poor. Although gemcitabine‐based chemotherapy is typically offered as the standard care, most patients do not survive longer than 6 months. Therefore, new therapeutic approaches are needed. The α‐gal epitope (Galα1‐3Galβ1‐4GlcNAc‐R) is abundantly synthesized from glycoproteins and glycolipids in non‐primate mammals and New World monkeys, but is absent in humans, apes and Old World monkeys. Instead, they produce anti‐Gal antibody (Ab) (forming approximately 1% of circulating immunoglobulins), which specifically interacts with α‐gal epitopes. Anti‐Gal Ab can be exploited in cancer immunotherapy as vaccines that target antigen‐presenting cells (APC) to increase their immunogenicity. Tumor cells or tumor cell membranes from pancreatic cancer are processed to express α‐gal epitopes. Subsequent vaccination with such processed cell membranes results in in vivo opsonization by anti‐Gal IgG in cancer patients. The interaction of the Fc portion of the vaccine‐bound anti‐Gal with Fcγ receptors of APC induces effective uptake of the vaccinating tumor cell membranes by the APC, followed by effective transport of the vaccinating tumor membranes to the regional lymph nodes, and processing and presentation of the tumor‐associated antigens. Activation of tumor‐specific B and T cells could elicit an immune response that in some patients is potent enough to eradicate the residual cancer cells that remain after completion of standard therapy. This review addresses these topics and new avenues of clinical importance related to this unique antigen/antibody system (α‐gal epitope/anti‐Gal Ab) and advances in immunotherapy in pancreatic cancer.     

Patients receiving murine monoclonal antibody therapy for malignancy develop T cells that proliferate in vitro in response to these antibodies as antigens

Peripheral blood mononuclear cells (PBMCs) were obtained from patients receiving radioactive murine monoclonal antibody (MAb) therapy for malignant epithelial tumours, as well as normal controls, and were tested for the ability of T cells to proliferate in vitro in the presence of the MAb administered for therapy (HMFG1), and another isotypically matched antibody of irrelevant specificity (11.4.1). We studied 13 patients who had one (ten patients) or two (three patients) courses of MAB treatment, 11 age matched patients with the same histologic types of tumours, that had not received MAbs, and four normal controls. There was a consistent dose dependent in vitro T cell proliferation in 11 of the 13 patients after MAb therapy. This was not observed in the pre-therapy group of patients or normal controls, where the T cell proliferative responses remained baseline. The mean stimulation index (S.I.) in the post-therapy group was significantly higher than that of the pre-therapy patients and that of normal controls. When the in vitro T cell proliferative responses of these patients were measured in the presence of HMGF1 MAb (IgG1) and an isotypically identical, but idiotypically unrelated 11.4.1 MAb (IgG1), there was no statistically significant difference in the mean S.I. For HMFG1 vs 11.4.1 for the whole group of treated patients. When patients were separated into those who received one and those who received two MAb treatments, a significant increase in the mean S.I. was observed in the presence of HMFG1, in the group of patients receiving two treatment courses, suggesting the generation of T cells with specificity for the idiotypic component of the administered murine immunoglobulin. In order to further characterise these in vitro cellular responses we incubated PBMCs with and without an optimal concentration of the MAb (100-300 micrograms ml-1), as defined by the proliferation assay, and compared the differences in cell subpopulations. A significant increase in the percentage of cells expressing interleukin-2 receptors (IL-2R) was observed after MAb stimulation. The percentage of CD4+ lymphocytes and the CD4/CD8 ratio increased in all the cases studied, after MAb stimulation, where the percentages of B cells and NK cells remained relatively constant at less than 2-3% of the total population. We therefore conclude that murine MAbs administered to patients with cancer can lead to the generation of T cells which can recognise these MAbs as antigens when presented appropriately in vitro. The main proliferating population appears to be T helper CD4+ lymphocytes which following stimulation can release interleukin-2 leading to the expression of high levels of IL-2R.