Rapid elimination of mouse/human chimeric monoclonal antibodies in nude mice

 At our laboratory we are currently evaluating the suitability of mouse/human chimeric monoclonal antibodies (cmAb) for use in radioimmunotherapy of patients with head and neck squamous cell carcinoma (HNSCC). We have developed cmAb containing the human constant IgG1 domain and the variable domains of murine mAb (mmAb) E48 and U36 respectively. We considered the tumour-bearing nude mouse to be a well-validated model for a first testing of the targeting capabilities of these cmAb in comparison with the mmAb. Therefore, 3 μg cmAb E48 (labelled with ¹²⁵I) and 3 μg mmAb E48 (labelled with ¹³¹I) were simultaneously injected into HNSCC-bearing nude mice and, at various assay times, mAb uptake in blood and other tissues was assessed. Remarkably, while in roughly 50% of the animals the biodistribution of the conjugates was similar, in the other animals cmAb E48 showed a much higher blood clearance than mmAb E48. This resulted in a lower tumour uptake of cmAb E48 in comparison with mmAb E48. To determine whether this phenomenon was related to mAb E48 or to the animal model, other cmAb-mmAb combinations were evaluated in the same way: cmAbs SF-25, 17-1A and U36 (all IgG1) were tested and all showed a rapid elimination in about 50% of the animals. Besides a decrease in blood concentration, an increase of cmAb levels in liver and spleen was observed within 24 h after injection. Isotype-specific enzyme-linked immunosorbent assays showed that mice that demonstrated a rapid elimination of cmAb from the blood had much lower endogenous IgG1, IgG2b and IgG3 titres than mice showing normal clearance. IgG2a levels were low in all mice. Biodistribution experiments with 3 μg chimeric 17-1A isoforms showed high blood clearance in a proportion of the mice for IgG1, IgG3 and IgG4, but not for IgG2. Increase of the cmAb dose to 100 μg resulted in a similar cmAb and mmAb biodistribution in all mice. Moreover, the biodistribution of the F(ab′)₂ fragment of an IgG1 cmAb was similar for all mice in contrast to that of coinjected whole IgG. On the basis of these results it can be hypothesized that, in mice with low endogenous IgG titres, cmAb with specific isotypes are rapidly removed from the blood (and ultimately from the body) by mediation of Fc-binding receptors. Apparently, in mice with high endogenous IgG titres or in mice receiving a high cmAb dose, these receptors are saturated. Furthermore, the rapid elimination of cmAb from nude mice, which may occur after injection at a low dose, is a phenomenon related to the nude mouse model. Received: 26 July 1996 / Accepted: 16 January 1997     

Granulocyte/macrophage-colony-stimulating factor augments the induction of antibodies,especially anti-idiotypic antibodies,to therapeutic monoclonal antibodies

A group of 86 patients with advanced colorectal carcinoma were treated with the mouse (m) (IgG2A) or chimeric (c) monoclonal antibody (mAb) 17-1A. Prior to therapy, no patient had detectable levels of antibodies to mAb17-1A. All mmAb17-1A-treated patients (n=76) developed antibodies against both idiotypic and isotypic determinants. Addition of granulocyte/macrophage-colony-stimulating factor (GM-CSF) to mmAb17-1A significantly enhanced the induction of anti-idiotypic (ab₂) as well as anti-isotypic antibodies. Of the mmAb17-1A-treated patients, 16 developed type I allergic reactions. These patients had significantly higher concentrations of anti-(mouse Ig) antibodies than patients without type I reactions. Of these 16 patients, 5 had received mmAb17-1A alone; they constituted 9% of this group (5/56). The remaining 11 patients had been given mmAb17-1A together with GM-CSF, and represented 55% of this treatment group (11/20). The difference was statistically significant (P<0.001). Of 10 patients, 9 (90%) treated with cmAb17-1A and GM-CSF developed ab₂. The ab₂ concentration in this patient group was significantly lower compared to those treated with mmAb-17A. Anti-(mouse Ig) antibodies caused clinical symptoms requiring therapeutic intervention in fewer than 10% of the patients treated with mmAb17-1A alone. With the addition of GM-CSF, the antibody concentration as well as the frequency of allergic side-effects calling for medical action increased significantly. Significantly more patients with a high ab₂ concentration (at least 15g/ml) 1 month after completion of mAb therapy responded to mAb treatment as compared to those with a low ab₂ concentration (P<0.05). Moreover, patients with a high ab₂ concentration (at least 15 g/ml) had a median survival time of 15 months while those with a lower concentration survived for a median time of 9 months (P=0.01).     

Humoral anti-idiotypic and anti-anti-idiotypic immune response in cancer patients treated with monoclonal antibody 17-1A

 A group of 96 patients with advanced colorectal carcinoma were treated with the mouse (m) or chimeric (c) (mouse variable regions × human IgG1 constant regions) monoclonal antibody (mAb) 17-1A recognizing the tumour-associated antigen GA733-2. Eighty-two of the 83 patients treated with mmAb17-1A and 69% of the patients given cmAb17-1A (n = 13) developed anti-idiotypic antibodies (ab₂). Auto-antibodies binding to tumour cells expressing GA733-2 were found in 7% of the patients. In a further 38 patients (40%) antitumour-cell antibodies, i.e. anti-anti-idiotypic antibodies (ab₃), were induced by the mAb17-1A therapy. Patients with detectable ab₃ after treatment had significantly higher ab₂ levels than those not developing ab₃. Addition of granulocyte/macrophage-colony-stimulating factor (GM-CSF) to mmAb17-1A significantly enhanced the induction of ab₂ as well as induction of anti-anti-idiotypic antibodies (ab₃), compared to mmAb17-1A alone. Patients with a high increase in antitumour-cell antibodies (ab₃) induced by the therapy lived significantly longer than patients with no or a low level of induction of ab₃ (P = 0.016). The results indicate that induction of an idiotypic network response might be an important effector mechanism in mAb therapy. Received: 20 October 1995 / Accepted: 18 December 1995     

Pharmacokinetics and biodistribution of a light-chain-shuffled CC49 single-chain Fv antibody construct

Murine monoclonal antibodies to tumor-associated glycoprotein 72 (anti-TAG-72 mAb B72.3 and CC49) are among the most extensively studied mAb for immunotherapy of adenocarcinomas. They have been used clinically to localize primary and metastatic tumor sites; however, murine mAb generally induce potent human anti-(mouse antibody) responses. The immunogenicity of murine mAb can be minimized by genetic humanization of these antibodies, where non-human regions are replaced by the corresponding human sequences or complementary determining regions are grafted into the human framework regions. We have developed a humanized CC49 single-chain antibody construct (hu/muCC49 scFv) by replacing the murine CC49 variable light chain with the human subgroup IV germline variable light chain (Hum4 V_L). The major advantages of scFv molecules are their excellent penetration into the tumor tissue, rapid clearance rate, and much lower exposure to normal organs, especially bone marrow, than occur with intact antibody. The biochemical properties of hu/muCC49 scFv were compared to those of the murine CC49 scFv (muCC49 scFv). The association constants (K _a) for hu/muCC49 and muCC49 constructs were 1.1 × 10⁶ M⁻¹ and 1.4 × 10⁶ M⁻¹ respectively. Pharmacokinetic studies in mice showed similar rapid blood and whole-body clearance with a half-life of 6 min for both scFv. The biodistribution studies demonstrated equivalent tumor targeting to human colon carcinoma xenografts for muCC49 and hu/muCC49 scFv. These results indicate that the human variable light-chain subgroup IV can be used for the development of humanized or human immunoglobulin molecules potentially useful in both diagnostic and therapeutic applications with TAG-72-positive tumors. Received: 29 December 1999 / Accepted: 4 February 2000     

Phase I trial of chimeric (human-mouse) monoclonal antibody L6 in patients with non-small-cell lung,colon, and breast cancer

We report a single institution phase I trial of chimeric (mouse-human) monoclonal antibody (chL6) directed against a tumor-associated cell surface antigen expressed in non-small cell lung, colon, and breast cancer. The results of the study were contrasted with a previous trial of murine L6. ChL6 was administered intravenously to 18 patients with advanced cancer as a single, 4–16 infusion in doses ranging from 350 mg/m² to 700 mg/m². One patient received four weekly doses of 350 mg/m². Patients were followed for side effects, localization of antibody to tumor cells, pharmacokinetics and the development of antibodies against chL6. Side effects associated with treatment were chills, fever, and nausea, which lasted 24–48 hours. Platelet count and absolute leukocyte count fell immediately after treatment, but returned to pretreatment levels by day 7. Localization of chL6 to tumor cells in vivo was seen at 350 mg/m² and saturation at 700 mg/m² and 350 mg/m² per week×4. The pharmacokinetics of this antibody appeared similar to its murine analogue. Human antibodies against chL6 were detected in only 4 of 18 patients. These antibodies were directed against murine variable regent and their titers were lower than those occurring in most patients who received murine L6 in an earlier trial. No tumor reductions were seen. Chimeric L6 appears to be a suitable antibody for delivering anti-tumor agents because of its low immunogenicity and favorable in vivo tumor binding characteristics.     

Comparative biological properties of a recombinant chimeric anti-carcinoma mAb and a recombinant aglycosylated variant

Summary It has been demonstrated previously that the degree of glycosylation of a molecule may alter its pharmacokinetic properties and, in the case of an antibody, its metabolism and other biological properties. Transfectomas producing aglycosylated chimeric B72.3(1) pancarcinoma monoclonal antibody (mAb) were developed by introduction of the eukaryotic expression construct pECMgpB72.3 HuG1-agly, into SP2/0 murine myeloma cells producing the chimeric chain of mAb B72.3. After cell cloning, one subclone with the highest binding to the TAG-72-positive human colon carcinoma was designated mAb aGcB72.3, and its biological and biochemical properties were compared with those of the chimeric B72.3(1), designated mAb cB72.3. Polyacrylamide gel electrophoresis showed that under non-reducing conditions, the molecular masses of the aGcB72.3 and cB72.3 mAbs were 162 kDa and 166 kDa respectively. The heavy chain of mAb aGcB72.3 had a slightly faster mobility than that of cB72.3, while the mobility of the light chains of the two chimeric mAbs was similar. No difference was observed in the isoelectric points of either chimeric mAb. Liquid competition radioimmunoassays demonstrated that the aGcB72.3 and cB72.3 mAbs have comparable binding properties to TAG-72. These studies demonstrate that aglycosylation of the chimeric IgG1 mAb B72.3 at theCh2 domain, as has been shown for other mAbs [Dorai H., Mueller B., Reisfeld R. A., Gillies S. D. (1991) Hybridoma 10:211; Morrison S. L., Oi V. T. (1989) Adv Immunol 44:65], eliminates antibody-dependent cell-mediated cytotoxicity activity, but does not substantially alter affinity or plasma clearance in mice. These studies also demonstrate for the first time (a) no difference in plasma clearance of an aglycosylated and a chimeric mAb in a primate after i.v. inoculation; (b) a difference (P 0.05) in mice in the more rapid peritoneal clearance of a chimeric mAb versus an aglycosylated chimeric mAb; (c) higher (0.05 P 0.1) tumor:liver ratios at 24, 72 and 168 h using¹¹¹In-labeled aglycosylated chimeric mAb versus chimeric mAb. Since the liver is the major site of metastatic spread for most carcinomas, slight differences in tumor to normal liver ratios may be important in diagnostic applications. These studies thus indicate that comparative analyses of a novel recombinant construct (i.e., aglycosylated) and its standard chimeric counterpart require documentation in more than one system and are necessary if one is ultimately to define optimal recombinant/chimeric constructs for diagnosis and therapy in humans.     

Differences in biodistribution of the anti-(carcinoembryonic antigen) murine monoclonal antibody CE-25, its F(ab′)2 fragment and its intact mainly human chimeric form CE 4-8-13

The effect of the size of the tumour and the amount of antibody injected on the biodistribution of a family of radioiodinated antibodies was studied. The intact mouse anti-(carcinoembryonic antigen) (anti-CEA) monoclonal antibody CE-25, its F(ab)₂ fragment and the intact human-mouse chimeric from CE 4-8-13 were evaluated in a model system using the human CEA-producing colon xenograft T 380 grown in nude mice. The relative retention (the percentage of the injected dose per gram of tissue), of mouse mAb and F(ab)₂ in tumour and most normal tissues 1 day after injection was independent of the antibody dose; after 4 days the mAb values increased with increasing antibody dose. The relative retention of chimeric mAb increased with increasing antibody dose 1 day after injection and also slightly after 4 days. The relative retention in tumour tissue was lower in bigger xenografts for all antibodies. The relative retention of mouse mAb in small tumours increased from day 1 to day 4; for chimeric mAb this value decreased. In normal tissues the relative retention of mouse mAb decreased from day 1 to day 4, but the relative retention of chimeric mAb in normal tissue dropped rapidly and changed little afterwards. Thus the biokinetics of antibodies is species-dependent: foreign, mainly human, chimeric antibody clears faster from normal mouse tissue than mouse antibody and reaches lower concentrations.     

Interaction of Human Ribosomal Protein S26 with Fragments of Its Own mRNA and Pre-mRNA in Nuclear Extract of HeLa Cells

As shown by nitrocellulose filtration assays with RNA fragments transcribed from various regions of the human ribosomal protein (rp) S26 gene, recombinant rpS26 binds to the first intron of the rpS26 pre-mRNA (apparent association constant (K _a) 5.0 · 10⁷ M^–1) and, to a lesser extent, to the rpS26 mRNA (K _a 2.0 · 10⁷ M^–1). The binding was specific, since human rpS19 had an order of magnitude lower K _a with the first intron and did not bind with the rpS26 mRNA. Immunoassays with specific antibodies showed that rpS26 contained in the nuclear extract of HeLa cells binds to the first intron of its pre-mRNA and, less efficiently, to its mRNA. In either case, RNA binding substantially increased in the presence of recombinant rpS26. Along with other (48K, 59K) nuclear proteins, rpS26 was assumed to form complexes, the functional role of which is storage of pre-mRNAs inactive in splicing.     

A human/mouse chimeric monoclonal antibody against CA125 for radioimmunoimaging of ovarian cancer

Murine monoclonal antibody 196-14 recognizes the ovarian-cancer-associated antigen CA 125, but the epitope it recognizes is different from that of monoclonal antibody OC125. We developed a human/mouse chimeric 196-14 using the variable regions of the murine 196-14 and human heavy-chain (l) and light-chain () constant regions. Cell binding and competitive inhibition assays using chimeric 196-14 labeled with¹²⁵I,¹¹¹In or^99mTc demonstrated that the in vitro immunoreactivity of the chimeric antibody was identical to that of the parental murine monoclonal antibody. However, in mice bearing human ovarian cancer xenografts, the clearance from blood was faster and absolute levels of accumulation in the tumor were lower for the¹²⁵I-labeled or^99mTc-labeled chimeric antibody than for the murine antibody labeled with the corresponding radionuclides. The tumor-to-blood radioactivity ratio was not significantly different between the chimeric antibody and the murine antibody, regardless of the radionuclide used for labeling. Chimeric antibody 196-14 labeled with¹³¹I,¹¹¹In or^99mTc is promising for the radioimmunoimaging of ovarian cancer.     

Functional properties of FC-2.15, a monoclonal antibody that mediates human complement cytotoxicity against breast cancer cells

FC-2.15 is a murine IgM monoclonal antibody (mAb) that recognizes a cell-surface antigen (Ag2.15) expressed in most tumor-proliferating cells of human breast carcinomas and other neoplasias. In this study the cytotoxic ability of mAb FC-2.15, its cell-surface binding properties and endocytosis in Ag2.15-expressing (Ag2.15⁺) cells were investigated. A⁵¹Cr-release assay was used to test the FC-2.15-mediated cytotoxicity. When human serum was used as source of complement, FC-2.15 exerted a strong cytotoxic effect against human Ag2.15⁺ cells such as MCF-7 (breast cancer cell line), primary breast carcinoma cells, polymorphonuclear leukocytes and chronic myeloid leukemia cells. The mAb concentration range was 1–50 g/ml. Cytotoxicity was completely abolished when complement was inactivated. Only 3.8±2.9% of MCF-7 cells survived the treatment with FC-2.15 in the presence of human serum. A flow-cytometry assay was performed to study the Ag2.15 expression of the surviving cells and they were found to be Ag2.15^–. FC-2.15 did not mediate antibody-dependent cell cytotoxicity when different effector cells were used. Scatchard analysis with¹²⁵I-FC-2.15 on MCF-7 cells demonstrated an affinity constant of 6.9×10⁷ M^–1 and 2.8×10⁶ antigenic sites/cell.¹²⁵I-FC-2.15 was internalized to cytoplasmic vesicles reaching a maximum of 27% after 6 h incubation, followed by the release of labeled degradation products to the supernatant. FC-2.15 appears to exert its cytotoxic effect mainly in the presence of human complement, it reacts with intermediate affinity with a high-density surface antigen, and it is slowly internalized by Ag2.15⁺ cells.     

Anti-idiotypic monoclonal antibody to a T-cell chronic lymphatic leukemia

Summary A murine anti-idiotypic monoclonal antibody (mAb), F1, (IgG_2a) was produced against the variable part of the T-cell receptor for antigen (T_i, /) on the tumor cells of a patient with T-cell chronic lymphatic leukemia (CD3⁺, 8⁺, 4^–). The molecular weight of the protein reactive with mAb F1, comodulation and coprecipitation with anti-CD3 antibody, and the restricted tumor-cell reactivity strongly support the anti-idiotypic nature of mAb F1. MAb F1 also stained 4% of peripheral blood lymphocytes of healthy donors. MAb F1 did not stimulate the tumor cells to DNA synthesis, but stimulated a fraction of the normal peripheral blood lymphocytes, mAb F1 did not mediate antibody-dependent cellular cytotoxicity or complement lysis to any significant degree in vitro. Three infusions of 1–10 mg anti-idiotypic mAb were given over a period of 4 weeks. The plasma half-life for mAb F1 was 3 h in the first 2 h after infusion and 44 h from 2 h to 120 h after infusion. After each treatment a rapid decrease of circulating tumor cells was seen. During the observation period an 80% reduction of the total circulating tumor cells was noted. After the second infusion, IgM and IgG antimouse antibodies were detected. Side-effects from therapy were fever, chills, nausea, vomiting, diarrhea, tachycardia, increase in systolic blood pressure and shortness of breath. Thus, in T-cell malignancies a major reduction of circulating tumor cells can be accomplished by low doses of anti-idiotypic mAb. Anti-idiotypic mAb might be a therapeutic agent of significant importance.     

Dual induction of apoptotic and autophagic cell death by targeting survivin in head neck squamous cell carcinoma

Survivin is ubiquitously expressed in patients with head neck squamous cell carcinoma (HNSCC) and is associated with poor survival and chemotherapy resistance. Sepantronium bromide (YM155) is a selective survivin suppressant that exhibits potent antitumor activities by inducing apoptosis and autophagy in various types of cancer. However, the curative effects and underlying mechanisms of YM155 in HNSCC remain unclear. This study showed that survivin overexpression positively correlated with p-S6, p-Rb and LAMP2 but negatively correlated with the autophagic marker LC3 in human HNSCC tissues. In vitro studies revealed that YM155 triggered apoptosis of HNSCC cells in mitochondria and death receptor-dependent manner. The treatment also significantly enhanced autophagy by upregulating Beclin1, which led to cell death. YM155 not only downregulated the expression of survivin but also remarkably suppressed the activation of the mTOR signaling pathway in vitro and in vivo. YM155 displayed potent antitumor activities in both CAL27 xenograft and transgenic HNSCC mice models by delaying tumor onset and suppressing tumor growth. Furthermore, YM155 combined with docetaxel promoted tumor regression better than either treatment alone without causing considerable body weight loss in the HNSCC xenograft models. Overall, targeting survivin by YM155 can benefit HNSCC therapy by increasing apoptotic and autophagic cell death, and suppressing prosurvival pathways.Head and neck squamous cell carcinoma (HNSCC), which occurs in the oral cavity, oropharynx, larynx and hypopharynx, is the sixth most common malignancy worldwide.¹ It affects 600 000 new patients each year, which accounts for over 90% of head and neck cancers.^{2, 3} The current preferred therapy for HNSCC is combined surgery, radiotherapy, chemotherapy and biotherapy; however, the 5-year survival rate is still <50%, and the long-term survival rate has only marginally improved.^{4, 5, 6} As an important hallmark of head and neck cancer, apoptosis resistance restricts the efficacy of traditional therapies.⁷ Survivin (also called BIRC5) inhibits apoptosis-related proteins, regulates cell division, relates to stress response and promotes tumor-associated angiogenesis in HNSCC.⁸ Survivin is also associated with high-grade and advanced HNSCC, poor survival, high recurrence rate and chemotherapy and radiation resistance. Therefore, targeting survivin is promisingly beneficial for head and neck cancer therapies.Sepantronium bromide (YM155) is a small imidazolium-based compound (1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide) that selectively suppresses survivin expression⁹ and displays potent anticancer activities against various types of cancer.^{10, 11, 12} Previous researches have focused on that YM155 induced the apoptosis by downregulating survivin in cancer cells.^{10, 13, 14} Recent studies including ours have demonstrated YM155 also triggered autophagy in cancer cells.^{15, 16, 17} Macroautophagy or autophagy is considered to be another type of programmed cell death wherein proteins are degraded by autophagosomes and lysosomes.¹⁸ Autophagy also has an important role in tumorigenesis.¹⁹ Autophagy shares several regulatory systems and common pathways with apoptosis; thus, autophagy is closely linked with apoptosis. Beclin1 (ATG6), an autophagy-specific gene that is essential for autophagosome induction and elongation, interacts with several apoptosis-related genes, such as bcl-2, bcl-xl and survivin.²⁰ Therefore, YM155 may not only induce the apoptosis but also affect the autophagy in HNSCC.The present study investigated the antitumor effects of YM155 on HNSCC in vitro and in vivo through dual induction of apoptotic and autophagic cell death. Although it specifically suppressed the expression of survivin, we here proved YM155 also targeted the mTOR signaling pathway, which was the principal regulator of cancer cell survival and autophagy. Most importantly, in an inducible tissue-specific spontaneous HNSCC mouse model with 100% penetrance by the combined deletion of Tgfbr1 and Pten (Tgfbr1/Pten 2cKO) in the oral mucosa²¹ with ubiquitous activation of the Akt/mTOR/survivin pathway,²² YM155 exerted significant therapeutic effects by delaying tumor onset and suppressing tumor growth. This finding coincided with the xenograft results. Finally, the effects of YM155 when combined with traditional chemotherapeutic agent were also determined.     

A mouse/human chimeric anti-(ganglioside GD3) antibody with enhanced antitumor activities

Ganglioside GD3, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin has been focused on as a target molecule for passive immunotherapy. We have cloned the cDNA encoding the immunoglobulin light and heavy chains of an anti-GD3 monoclonal antibody KM641 (murine IgG3, ), and constructed the chimeric genes by linking the cDNA fragments of the murine light and heavy variable regions to cDNA fragments of the human and 1 constant regions, respectively. The transfer of these cDNA constructs into SP2/0 mouse myeloma cells resulted in the production of the chimeric antibody, designated KM871, that retained specific binding activity to GD3. Indirect immunofluorescence revealed the same staining pattern for chimeric KM871 and the mouse counterpart KM641 on GD3-expressing melanoma cells. When human serum and human peripheral blood mononuclear cells were used as effectors in complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity respectively, the chimeric KM871 was more effective in killing GD3-expressing tumor cells than was the mouse counterpart KM641. Intravenous injection of chimeric KM871 markedly suppressed tumor growth in nude mice. The chimeric KM871, having enhanced antitumor activities and less immunogenicity than the mouse counterpart, would be a useful agent for passive immunotherapy of human cancer.     

Minor human antibody response to a mouse and chimeric monoclonal antibody after a single i.v. infusion in ovarian carcinoma patients: a comparison of five assays

The human anti-(mouse Ig) antibody (HAMA) response was measured in serum of 52 patients suspected of having ovarian carcinoma who had received an i.v. injection of either the murine monoclonal antibody (mAb) OV-TL 3 F(ab)₂ (n=28, 1 mg) or the chimeric mouse/human mAb MOv18 (cMOv18;n=24, 3 mg). Serum samples were taken before injection and 2–3 and 6–14 weeks after administration. A double-antigen or bridging assay was developed to detect responses against both murine as well as chimeric antibodies. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) as well as three commercially available assays were used to study antibody response against the murine antibody OV-TL 3. With both the double-antigen (bridging) assay and the indirect ELISA 1 of the 28 patients (4%) injected with murine OV-TL 3 F(ab)₂ showed a HAMA reaction 6 weeks after injection, which was demonstrated to be a mixed anti-isotypic and anti-idiotypic response. None of the 24 patients injected with the chimeric MOv18 showed an anti-chimeric antibody response. The various commercially available assays demonstrated conflicting results. The double-antigen-or bridging assay is a reliable method to detect anti-murine and antichimeric antibodies. The assay can be easily adapted for use with human antibodies. The immunogenicity of OV-TL 3 F(ab)₂ and cMOv18 in patients is low, making both antibodies candidates for immunotherapy.This work was supported by a clinical research grant of the Netherlands Organization for Scientific Research (NWO 900-716-020) and by the Biocare Foundation (grant 92-05).     

Development of an Enzyme-Linked Immunosorbent Assay System for the Determination of Asymmetric Dimethylarginine Using a Specific Monoclonal Antibody

We produced a monoclonal antibody (mAb) against N ^G,N ^G-dimethyl-L-arginine (asymmetric dimethylarginine: ADMA), an endogenous competitive inhibitor of nitric oxide synthase (NOS), and developed an enzyme-linked immunosorbent assay (ELISA). The competitive ELISA method using the mAb determined 5 nM–100 nM ADMA, and ADMA levels in human plasma and urine were found to be 0.78 μM and 51.3 μmol/g of creatinine respectively.     

The comparative effects of IL-1 and TNF on AMN-anti-Ly 2.1 immunoconjugate therapy

The administration of mTNF and hIL-1 was investigated for their potential to increase the anti-tumor activity of AMN-anti-Ly-2.1 against the Ly-2.1⁺ murine thymoma ITT(1) 75 NS E3. Dose response studies using mTNF alone demonstrated a single 10g iv injection produced 30% inhibition in tumor size while 3 doses of 1g administered on alternative days produced 70% tumor inhibition. By contrast, hIL-1 was unable to significantly reduce E3 tumor size using single doses up to 10g or a total of 30g administered in 3 doses (iv or ip). However, intratumor injection of hIL-1 (20g injected in 2 doses) produced 20% inhibition in tumor size. Combination therapy using AMN immunoconjugates with mTNF showed enhanced antitumor activity compared to each agent alone. Biodistribution studies revealed that anti-tumor activity, was due to increased localization (2–3 fold) of AMN immunoconjugates in the presence of mTNF- whereas huIL-1 was without effect unless accompanying toxicity was seen. Clearly for this tumor, mTNF potentiated the effects of AMN immunoconjugates. Despite the shared biological properties of these cytokines, mTNF is superior to hIL- for augmenting drug immunoconjugate.Abbreviations AMN Aminopterin - CBF₁ (C57BL6xBALB/c)F₁ mice - DMSO Dimethyl sulfoxide - E3 ITT(1)75NS E3 - HAMA human-anti-mouse antibody - i.p. intraperitoneal(ly) - I.T. intratumor - i.v. intravenous(ly) - hIL-1 recombinant human Interleukin-1-alpha - MoAb monoclonal antibody - PBS phosphate buffered saline - SE standard error - s.c. subcutaneous(ly) - mTNF recombinant murine tumor necrosis factor-alpha     

Antibody-dependent difference in biodistribution of monoclonal antibodies in animal models and humans

 The study was designed to clarify the difference in pharmacokinetics of monoclonal antibodies (mAb) in animal models and humans, and to elucidate the applicability of animal models. ^99mTc-labeled murine mAb – against carcinoembryonic antigen (designated BW431/26), and neural cell adhesion molecule (NE150) – and one chimeric mouse/human mAb against nonspecific cross-reacting antigen (chNCA) were administered i.v. to normal mice and athymic mice (370 kBq, 400 ng) xenografted with human cancer cells expressing antigens, and into patients with tumor (925 MBq, 1 mg). The biodistribution of two of the three mAb (not ^99mTc-BW431/26) differed clearly in mice and patients. ^99mTc-NE150 showed specific uptake in xenografted tumor and otherwise a normal biodistribution; however, clinical examination showed increased uptake in the liver with rapid blood clearance (mean α half-life = 31.1 min) compared with ^99mTc-BW431/26 (28.4 h). ^99mTc-chNCA demonstrated increased blood clearance and renal excretion in both normal and athymic mice, with accumulation in tumors. Clinical examination showed rapid blood clearance (mean α half-life = 6.4 min) and increased uptake in the liver. High-performance liquid chromatographic analysis of ^99mTc-chNCA revealed the immune complex in blood, suggesting uptake of the complex by the reticuloendothelial cells. The biodistribution of radiolabeled mAb in animal and human models was variable and specific for each of the three mAb. The results of animal studies with mAb should be evaluated carefully before being extrapolated to humans, on the basis of the nature of the mAb and interacting substances. Received: 9 April 1997 / Accepted 3 March 1998     

Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies

The HER2 protooncogene encodes a receptor tyrosine kinase, p185^HER2. The overexpression of p185^HER2 has been associated with a worsened prognosis in certain human cancers. In the present work we have screened a variety of different tumor cell lines for p185^HER2 expression using both enzyme-linked immunosorbent and fluorescence-activated cell sorting assays employing murine monoclonal antibodies directed against the extracellular domain of the receptor. Increased levels of p185^HER2 were found in breast (5/9), ovarian (1/6), stomach (2/3) and colorectal (5/16) carcinomas, whereas all kidney and submaxillary adenocarcinoma cell lines tested were negative. Some monoclonal antibodies directed against the extracellular domain of p185^HER2 inhibited growth in monolayer culture of breast and ovarian tumor cell lines overexpressing p185^HER2, but had no effect on the growth of colon or gastric adenocarcinomas expressing increased levels of this receptor. The most potent growth-inhibitory anti-p185^HER2 monoclonal antibody in monolayer culture, designated mumAb 4D5 (a murine IgG1 antibody), was also tested in soft-agar growth assays for activity against p185^HER2-overexpressing tumor cell lines of each type, with similar results. In order to increase the spectrum of tumor types potentially susceptible to monoclonal antibody-mediated anti-p185^HER2 therapies, to decrease potential immunogenicity issues with the use of murine monoclonal antibodies for human therapy, and to provide the potential for antibody-mediated cytotoxic activity, a mouse/human chimeric 4D5 (chmAb 4D5) and a humanized 4D5 (rhu)mAb 4D5 HER2 antibody were constructed. Both engineered antibodies, in combination with human peripheral blood mononuclear cells, elicited antibody-dependent cytotoxic responses in accordance with the level of p185^HER2 expression. Since this cytotoxic activity is independent of sensitivity to mumAb 4D5, the engineered monoclonal antibodies expand the potential target population for antibody-mediated therapy of human cancers characterized by the overexpression of p185^HER2.     

Somatic embryogenesis and plantlet regeneration from cell suspension cultures of palmarosa grass (Cymbopogon martinii)

A cell suspension culture was established from nodal callus ofCymbopogon martinii (Roxb.) Wats in a liquid medium containing Murashige and Skoog (1962) basal salts, vitamins, 100 mg 1^–1 myo-inositol and 20 g l^–1 of sucrose (MS) that was supplemented with 13.6 M 2,4-dichlorophenoxyacetic acid and 1.15 M kinetin. An initial inoculum density of 2 x 10⁴ cells ml^–1exhibited optimum cell growth. Calli were obtained 12–15 days after the suspension was plated onto semisolid medium of a similar composition. When calli were transferred to semisolid regeneration medium containing MS + 6.7 M N ⁶-benzyl-adenine + 1.15 M kinetin, somatic embryogenesis and plantlet regeneration occurred after 10–25 days. There was no significant decrease in the regeneration potential of the calli even when the cultures were initiated from 47-week-old cell suspensions. Chromosome counts of cells in suspensions, calli and somatic embryos derived from cultures of different ages revealed the presence of diploids, tetraploids and octaploids. However, the 33 regenerated plants tested were all diploid, indicating that only diploid cells were capable of regeneration in vitro.Abbreviations MS Murashige and Skoog (1962) basal salts with vitamins (100 mg1^–1 myo-inositol, 20 g1^–1 sucrose) - 2,4-D 2,4-dichlorophenoxyacetic acid - BA N ⁶-benzyl-adenine - Kn kinetin - MSC MS + 13.6 M 2,4-D + 1.15 M Kn - MSR MS +6.7 M BA + 1.15 M Kn     

Analytical techniques for boron and boron 10 analysis in a solid experimental tumor EO. 771

Summary If a tumor can be preferentially loaded with a suitable boron-10 compound and irradiated with thermal neutrons, malignant cells can be selectively destroyed via the-particle + Li 7-nucleus from the reaction¹⁰B(n,)⁷Li.Neutron capture therapy with two boron-10 amino acid analogs of low toxicity has been tested in recent years: (a) trimethylamine-carboxyborane, (A3) and (b) amine-carboxyborane, (A7). Now the boron-10 glycineamide analog (A8), amineboryl-carboxamide has been synthesized; it contains 13.81% boron (90% Boron 10+10% Boron 11) and shows a very low toxicity in mice. The effects of this compound were tested on the syngeneic solid adenocarcinoma EO 771 on the right hind leg of male C57 BL/6J mice under standard conditions, by measuring tumor volume growth delay and cell cycle changes using flow cytometry. Boron distribution between tumor and muscle was analyzed by emission spectroscopy with inductively coupled plasma (ICP) following injection of a suspension of peanut oil emulsion. In addition, boron-10 concentration in the tumor were analyzed with prompt-activation analysis and neutron capture radiography (Kodak-Pathé LR 115) at the MRR reactor in Brookhaven after i.p. injection of 0.4 mg/g A8.Application of A8 alone (0.4 mg/g i.p.) or thermal neutron irradiation of the tumor EO. 771 produced a tumor growth delay of 1–2 days for tumor volume doubling. Application of the boron 10 glycine-amide analog A8 i.p. plus 510¹² n/cm² resulted in a growth delay of 3–6 days.In contrastintratumoral application of A8 plus 410¹² n/cm² neutrons gave a growth delay of 7–14 days; the fraction of (G2 + M) cells rose from 35% (neutrons alone) to 52%, as evaluated from flow cytometry.Dedicated to Prof. L.E. Feinendegen on the occasion of his 60th birthday