90Y-Labeled Anti-ROBO1 Monoclonal Antibody Exhibits Antitumor Activity against Small Cell Lung Cancer Xenografts

IntroductionROBO1 is a membrane protein that contributes to tumor metastasis and angiogenesis. We previously reported that ⁹⁰Y-labeled anti-ROBO1 monoclonal antibody (⁹⁰Y-anti-ROBO1 IgG) showed an antitumor effect against ROBO1-positive tumors. In this study, we performed a biodistribution study and radioimmunotherapy (RIT) against ROBO1-positive small cell lung cancer (SCLC) models.MethodsFor the biodistribution study, ¹¹¹In-labeled anti-ROBO1 monoclonal antibody (¹¹¹In-anti-ROBO1 IgG) was injected into ROBO1-positive SCLC xenograft mice via the tail vein. To evaluate antitumor effects, an RIT study was performed, and SCLC xenograft mice were treated with ⁹⁰Y-anti-ROBO1 IgG. Tumor volume and body weight were periodically measured throughout the experiments. The tumors and organs of mice were then collected, and a pathological analysis was carried out.ResultsAs a result of the biodistribution study, we observed tumor uptake of ¹¹¹In-anti-ROBO1 IgG. The liver, kidney, spleen, and lung showed comparably high accumulation of ¹¹¹In-labeled anti-ROBO1. In the RIT study, ⁹⁰Y-anti-ROBO1 IgG significantly reduced tumor volume compared with baseline. Pathological analyses of tumors revealed coagulation necrosis and fatal degeneration of tumor cells, significant reduction in the number of Ki-67-positive cells, and an increase in the number of apoptotic cells. A transient reduction of hematopoietic cells was observed in the spleen, sternum, and femur.ConclusionsThese results suggest that RIT with ⁹⁰Y-anti-ROBO1 IgG is a promising treatment for ROBO1-positive SCLC.     

Correlation of beta-camera imaging and immunohistochemistry in radioimmunotherapy using90Y-labeled monoclonal antibodies in ovarian cancer animal models

Tumor stroma contains much fibrin and monoclonal antifibrin antibody targeting is possible in tumors. In this study, nude mouse human ovarian carcinoma xenograft specimens were investigated after treatment with⁹⁰Y-labeled monoclonal antifibrin antibody Fab fragment or with⁹⁰Y-labeled OC125-monoclonal antibody F(ab′)₂ fragments. The mice received the radioimmunotherapy activity either intratumorally, intraperitoneally, or intravenously. Beta-camera imaging (BCI) is a novel device for studying activity distribution in tissue specimens and, together with immunohistochemistry (IHC) with OC125, antifibrin, anticarcinoembryonic antigen, anti-cytokeratin, and anti-placental alkaline phosphatase antibodies, was used for correlation of activity distribution of tissue specimens. These results were in concordance: Antigen distribution measured with IHC and radioactivity distribution were similar with the same antibodies, antifibrin, and OC125: However, these antigens demonstrated rather different distribution. Tissue studies revealed that activity was concentrated also in the necrotic tumor tissue, indicating that cell death was also caused by radiation. Differences in the tumor cell morphology were observed using different routes of administration. With BCI, it is possible to quantitate activities in frozen sections (microdosimetry), and these results were in concordance with absolute activities as measured by tissue sampling and well-counting. Three-dimensional reconstruction of tissue slices combined with radioactivity distribution measured with BCI allows estimation of total absorbed radiation dose in tumor after an appropriate dose planning.     

Preclinical assessment of 90Y-labeled monoclonal antibody Co17-1A,a potential agent for radioimmunotherapy of colorectal carcinoma

Monoclonal antibody CO17-1A was radiolabeled with ⁹⁰Y using the cyclic DTPA anhydride technique and administered intravenously to athymic nude mice bearing SW 948 human colorectal carcinomas. The tumor specificity of ⁹⁰Y-CO17-1A was improved by coadministration of 100 μg of the unlabeled antibody per animal and by purification using HPLC instead of column gel filtration chromatography. Absorbed radiation dose estimates for ⁹⁰Y-CO17-1A were calculated. The radiation dose to the bone marrow will limit the amount of ⁹⁰Y-CO17-1A that can be administered for cancer therapy.     

Physiologically Based Pharmacokinetic Modeling Is Essential in 90Y-Labeled Anti-CD66 Radioimmunotherapy

IntroductionRadioimmunotherapy (RIT) with ⁹⁰Y-labeled anti-CD66 antibody is used to selectively irradiate the red marrow (RM) before blood stem cell transplantation of acute leukemia patients. To calculate the activity to administer, time-integrated activity coefficients are required. These are estimated prior to therapy using gamma camera and serum measurements after injection of ¹¹¹In labeled anti-CD66 antibody. Equal pre-therapeutic and therapeutic biodistributions are usually assumed to calculate the coefficients. However, additional measurements during therapy had shown that this assumption had to be abandoned. A physiologically based pharmacokinetic (PBPK) model was developed to allow the prediction of therapeutic time-integrated activity coefficients in eight patients.AimsThe aims of the study were to demonstrate using a larger patient group 1) the need to perform patient-specific dosimetry in ⁹⁰Y-labeled anti-CD66 RIT, 2) that pre-therapeutic and therapeutic biodistributions differ, and most importantly 3) that this difference in biodistributions can be accurately predicted using a refined model.ResultsVariability of the RM time-integrated activity coefficients ((37.3±7.5) h) indicates the need for patient-specific dosimetry. The relative differences between pre-therapeutic and therapeutic serum time-activity curves were (-25±16)%. The prediction accuracy of these differences using the refined PBPK models was (-3±20)%.ConclusionIndividual treatment is needed due to biological differences between patients in RIT with ⁹⁰Y-labeled anti-CD66 antibody. Differences in pre-therapeutic and therapeutic biokinetics are predominantly caused by different degrees of saturation due to different amounts of administered antibody. These differences could be predicted using the PBPK models.     

The natural sulfoglycolipid derivative SQAP improves the therapeutic efficacy of tissue factor-targeted radioimmunotherapy in the stroma-rich pancreatic cancer model BxPC-3

α-Sulfoquinovosylacyl-1,3-propanediol (SQAP) is a semi-synthetic derivative of natural sulfoglycolipid that sensitizes tumors to external-beam radiotherapy. How SQAP affects internal radiotherapy, however, is not known. Here, we investigated the effects of SQAP for radioimmunotherapy (RIT) targeting tissue factor (TF) in a stroma-rich refractory pancreatic cancer mouse model, BxPC-3. A low dose of SQAP (2 mg/kg) increased tumor uptake of the ¹¹¹In-labeled anti-TF antibody 1849, indicating increased tumor perfusion. The addition of SQAP enhanced the growth-inhibitory effect of ⁹⁰Y-labeled 1849 without leading to severe body weight changes, allowing for the dose of ⁹⁰Y-labeled 1849 to be reduced to half that when used alone. Histologic analysis revealed few necrotic and apoptotic cells, but Ki-67–positive proliferating cells and increased vascular formation were detected. These results suggest that the addition of a low dose of SQAP may improve the therapeutic efficacy of TF-targeted RIT by increasing tumor perfusion, even for stroma-rich refractory pancreatic cancer.     

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (K_A = 1.9 × 10⁵?1.4 × 10⁸ M^?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (K_A = 2.0 × 10⁵?1.8 × 10⁷ M^?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.     

Biodistribution and radioimmunotherapy of human breast cancer xenografts with radiometal-labeled DOTA conjugated anti-HER2/neu antibody 4D5

HER2/neu oncogene encodes a 185 kDa trans-membrane protein which is overexpressed in 20-30% of breast and ovarian cancers and portends a poor prognosis. We have studied the targeting and therapy of this oncoprotein with 4D5, a murine monoclonal antibody which recognizes a distinct epitope on the extracelluar domain of HER2/neu. We conjugated the antibody with an active ester of the macrocyclic chelating agent DOTA, radiolabeled the conjugate with either (111)In or (90)Y, and studied the antibody distribution and therapy, respectively, in athymic mice bearing xenografts of MCF7/HER2/neu, a human breast cancer cell line transfected with the HER2/neu oncogene. For the biodistribution of (111)In-labeled DOTA-4D5, a high specificity of tumor localization (30% ID/g) was seen with a tumor-to-blood ratio of greater than 2 at 48 h postinjection. Compared to a previously published study with (125)I-labeled 4D5 in beige nude mice bearing NIH3T3/HER2/neu xenografts [De Santes et al. (1992) Cancer Res. 52, 1916-1923], (111)In-labeled 4D5 antibody gave superior antibody uptake in tumor (30% ID/g vs 17% ID/g at 48h). In the therapy study, treatment of the nude mice bearing MCF7/HER2/neu xenografts with 100 microCi (3 microg) of (90)Y-labeled DOTA-4D5 caused a 3-fold reduction of tumor growth compared to untreated controls (injected with human serum albumin) in 40 days. Treatment of animals with 100 microCi of nonspecific antibody (90)Y-labeled DOTA-Leu16 (3 microg) had no tumor growth inhibition. Treatment with unlabeled DOTA-4D5 (3 microg) had a slight effect on tumor growth compared to untreated controls. When analyzed at the level of single animals, no effect was seen in seven of nine animals; however, in two of the animals, tumor growth inhibition was observed. Although a cold antibody therapeutic effect was unexpected at this dose level (3 microg), it may be possible that in some animals that 3 microg of antibody of (90)Y-labeled DOTA-4D5 augmented tumor growth reduction. To further explore the effects of cold antibody treatment alone, animals were treated with 100 or 400 microg of unlabeled 4D5 administered in two doses. These animals showed a 1.7-1.8-fold reduction in tumor growth over 28 days, a result less than that obtained with RIT only.     

Construction and biological activity of a recombinant bispecific single-chain antibody designed for therapy of minimal residual colorectal cancer

 Unlike monoclonal antibodies, clinical application of bispecific antibodies has so far lagged behind because of difficult, low-yield production techniques as well as considerable toxicity attributed to bispecific antibody preparations containing immunoglobulin-Fc parts and anti-CD3 homodimers [10, 2]. These difficulties were overcome by recombinant generation of a bispecific single-chain antibody (bscAb) joining two single-chain Fv fragments via a five-amino-acid glycine-serine linker. The anti-CD3 specificity directed against human T cells was combined with another specificity against the epithelial 17-1A antigen. The following domain arrangement was critical in this individual case to obtain a fully functional bscAb: VL_17-1A-VH_17-1A-VH_CD3-VL_CD3. The bscAb was expressed in chinese hamster ovary cells with a yield of 15 mg/l culture supernatant whereas numerous attempts to obtain a functional protein expression in Escherichia coli failed. The low-molecular-mass bispecific construct (60 kDa) could easily be purified by its C-terminal histidine tail. The antigen-binding properties are indistinguishable from those of the corresponding univalent single-chain Fv fragments as shown by enzyme immunoassay and flow cytometry. We could show that the bscAb, which lacks Fc parts and anti-CD3 homodimers is highly cytotoxic for 17-1A positive tumor cells in nanomolar concentrations and in the presence of human T cells. Most remarkably, it does not stimulate T lymphocyte proliferation in the absence of tumor cells and, moreover, does not induce CD25 up-regulation and the secretion of potentially toxic lymphokines such as tumor necrosis factor α, interleukin-6 and interferon γ. Maximal cytotoxicity (⁵¹Cr release) was achieved without notable costimulation and was not further enhanced by adding costimulatory signals such as those delivered by anti-CD28 antibodies. CD8⁺ as well as CD4⁺ T cell subpopulations were recruited to exert cytotoxicity against tumor cells with different kinetics. CD8⁺ cells induced high ⁵¹Cr release within 4 h while CD4⁺ cells required a 20-h incubation. The systemic application of the 17-1A/CD3-bscAb could be a major improvement in therapy against disseminated micrometastatic tumor cells. A prospective, randomized clinical trial showing that an anti-17-1A monoclonal antibody could prolong survival of colorectal cancer patients after 5 and 7 years, warrants an assessment of the clinical efficacy of this bscAb exhibiting a 1000-fold higher specific cytotoxicity against tumor cells in virto. Accepted: 14 October 1997     

Chemotherapy Synergizes with Radioimmunotherapy Targeting La Autoantigen in Tumors

Background

To date, inefficient delivery of therapeutic doses of radionuclides to solid tumors limits the clinical utility of radioimmunotherapy. We aim to test the therapeutic utility of Yttrium-90 (⁹⁰Y)-radio-conjugates of a monoclonal antibody, which we showed previously to bind specifically to the abundant intracellular La ribonucleoprotein revealed in dead tumor cells after DNA-damaging treatment.

Methodology/Principal Findings

Immunoconjugates of the DAB4 clone of the La-specific monoclonal antibody, APOMAB®, were prepared using the metal chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and then radiolabeled with ⁹⁰Y. Mice bearing established subcutaneous tumors were treated with ⁹⁰Y-DOTA-DAB4 alone or after chemotherapy. Non-radiosensitizing cyclophosphamide/etoposide chemotherapy was used for the syngeneic EL4 lymphoma model. Radiosensitizing cisplatin/gemcitabine chemotherapy was used for the syngeneic Lewis Lung carcinoma (LL2) model, and for the xenograft models of LNCaP prostatic carcinoma and Panc-1 pancreatic carcinoma. We demonstrate the safety, specificity, and efficacy of ⁹⁰Y-DOTA-DAB4-radioimmunotherapy alone or combined with chemotherapy. EL4 lymphoma-bearing mice either were cured at higher doses of radioimmunotherapy alone or lower doses of radioimmunotherapy in synergy with chemotherapy. Radioimmunotherapy alone was less effective in chemo- and radio-resistant carcinoma models. However, radioimmunotherapy synergized with radiosensitizing chemotherapy to retard significantly tumor regrowth and so prolong the survival of mice bearing LL2, LNCaP, or Panc-1 subcutaneous tumor implants.

Conclusions/Significance

We report proof-of-concept data supporting a unique form of radioimmunotherapy, which delivers bystander killing to viable cancer cells after targeting the universal cancer antigen, La, created by DNA-damaging treatment in neighboring dead cancer cells. Subsequently we propose that DAB4-targeted ionizing radiation induces additional cycles of tumor cell death, which further augments DAB4 binding to produce a tumor-lethal ‘genotoxic chain reaction’. Clinically, this approach may be useful as consolidation treatment after a drug-induced cell death among (small-volume) metastatic deposits, the commonest cause of cancer death.This article is part II of a two-part series providing proof-of-concept for the diagnostic and therapeutic use of the DAB4 clone of the La-specific monoclonal antibody, APOMAB®.     

Comparison of the biodistribution of 75Se- and 131I-labelled monoclonal antibodies in nude mice

A monoclonal antibody, C-215, against colon cancer, was internally labelled with [⁷⁵Se]methionine. The biodistribution was studied in tumour-bearing nude mice and compared with the biodistribution of [¹³¹I]C-215. The tissue uptake was divided into three parts: antibody bound to the antigen, antibody in the extracellular space and uptake of the released radionuclide. [⁷⁵Se]C-215 showed a greater amount of antigen-bound antibody in the tumour, but also a greater unspecific uptake both in tumour and normal tissue.     

In vivo therapeutic effect of CDH3/P-cadherin-targeting radioimmunotherapy

Purpose

We examined the possible efficacy of the yttrium-90 (⁹⁰Y)-labeled anti-CDH3/P-cadherin mouse monoclonal antibody (MAb-6) in radioimmunotherapy (RIT) for lung and colorectal cancers that express CDH3/P-cadherin.

Experimental design

MAb-6 was established using genetic immunization. The biodistribution of MAb-6 in nude mice with lung and colorectal cancers was examined by administering indium-111(¹¹¹In)-labeled MAb-6 to mice. The mice were prepared by inoculation of CDH3/P-cadherin-positive (EBC1, H1373, and SW948) and CDH3/P-cadherin-negative (A549 and RKO) tumor cells. Therapeutic effects and toxicity were investigated by administration of ⁹⁰Y-labeled MAb-6 (⁹⁰Y-MAb-6) to EBC, H1373, and SW948-inoculated mice.

Results

Our in vivo results confirmed the specific binding of MAb-6 to tumor cells after intravenous injections of ¹¹¹In-labeled MAb-6 to mice with tumors expressing CDH3/P-cadherin. A single intravenous injection of ⁹⁰Y-MAb-6 (100?μCi) significantly suppressed tumor growth in mice with tumors expressing CDH3/P-cadherin. Furthermore, two injections of ⁹⁰Y-MAb-6 led to complete tumor regression in H1373-inoculated mice without any detectable toxicity.

Conclusions

Our findings demonstrate that CDH3/P-cadherin-targeting RIT with ⁹⁰Y-MAb-6 is a promising strategy for the treatment for cancers expressing CDH3/P-cadherin.     

High-dose,unlabeled, nonspecific antibody pretreatment: influence on specific antibody localization to human melanoma xenografts

Summary Nonspecific uptake of radiolabeled monoclonal antibodies in normal tissues is a significant problem for tumor imaging. A potential means of decreasing nonspecific antibody binding is to blockade nonspecific antibody binding sites by predosing with cold, nonspecific isotypematched antibody, before injecting specific antibody. Nontumor-specific murine monoclonal antibody LK2H10 (IgG1) or Ab-1 (IgG2a) was given i.v. at doses of 0 to 3.5 mg to nude mice with xenografts of human melanoma. These mice were then given i.v. 4 g of ¹³¹I anti-high molecular weight antigen of melanoma (HMWMAA) monoclonal antibody 763.24T (IgG1) or 225.28S (IgG2a), respectively. These mice were also given a tracer dose of ¹²⁵I LK2H10 or Ab-1, respectively. Specific tumor uptake of anti-HMWMAA antibodies was see in all cases. No drop in tumor or nontumor uptake was demonstrated for either of the tumor-specific or nonspecific monoclonal antibodies due to nonspecific monoclonal antibody pretreatment. These data suggest that high doses of isotype-matched unlabeled nonspecific monoclonal antibody given before ¹³¹I tumor-specific monoclonal antibody, will not enhance tumor imaging. Present address: Hybritech, San Diego, CA, USA     

Preparation and immunological features of syngeneic monoclonal anti-idiotypic antibody using complementary determining region (CDR) peptide as the immunogen

A syngeneic monoclonal idiotypic antibody was prepared by immunizing the sequence peptide of complementary determining region-1 (CDRL-1) of 41S-2-L which is an antibody light chain capable of catalytically decomposing the antigen peptide (gp41 peptide:original antigen) as well as the intact gp41 molecule of HIV-1 envelope. The obtained idiotypic antibody, i41SL1-2, showed a high specificity to the CDRL-1 peptide. The intact i41SL1-2 and its heavy and light chains displayed apparent affinity constants to the CDRL-1 peptide of 3.6 × 10⁹, 2.7 × 10⁷, 1.8 × 10⁶/M, respectively. The i41SL1-2 recognized the artificial molecule CA2, which has a more complex steric conformation than the CDRL-1, while the i41SL1-2 showed very low affinity to the original monoclonal antibody 41S-2 and its light chain 41S-2-L. However, a homologous sequence, EGG-D, with the gp41 peptide was expressed in the complementary determining region-3 (CDRH-3) of the heavy chain of i41SL1-2. Furthermore, the consensus sequence EGG was located at the important position of the CDRH-3 loop of i41SL1-2. Although the sequence of CDRL-1 (16 mer) is quite shorter than that of whole light chain (112 mer), the CDRL-1 could induce the rearrangement of CDRH-3 gene of i41SL1-2 so as to express a homologous sequence with the original antigen.     

90Y Labeling of monoclonal antibody MOv18 and preclinical validation for radioimmunotherapy of human ovarian carcinomas

The monoclonal antibody (mAb) MOv18 binds the membrane alpha isoform of the folate receptor (FR) which is overexpressed in human ovarian carcinoma cells. Exploiting the targeting capacity of this mAb, we developed and preclinically validated a protocol for the stable labeling of the mAb with ⁹⁰Y, an isotope which has shown promise in cancer radioimmunotherapy. MOv18 was derivatized with the stable macrocyclic ligand p-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid (Bz-DOTA). MOv18-Bz-DOTA conjugates were labeled with ⁹⁰Y or ¹¹¹In under metal-free and good laboratory practice conditions. At the optimal Bz-DOTA/mAb derivatization ratio of 4–5, conjugates maintained binding activity up to 6 months, were efficiently labeled with ⁹⁰Y or ¹¹¹In (mean labeling yield 85 and 64%, associated to a final mean specific activity of 74 and 37 MBq/mg) and displayed a mean immunoreactivity of 60 and 58%, respectively. The radiolabeled preparations were stable in human serum, with >97% radioactivity associated to mAb at 48 h after labeling. The ability of ⁹⁰Y- and ¹¹¹In-MOv18 to localize FR on tumors in vivo was analyzed in nude mice bearing tumors induced by isogenic cell lines differing only in the presence or absence of the relevant antigen [A431FR (FR-positive) and A431tMock (FR-negative)]. In vivo biodistribution in organs other than tumor was comparable in non-tumor-, A431tMock- and A431FR-bearing mice, whereas the median tumor uptake of the radiolabeled reagents, expressed as area under the curve (AUC) and maximum uptake (U_max), was significantly higher (sixfold to sevenfold) in A431FR than in A431tMock tumors (P=0.0465 and P=0.0332, respectively). Mean maximum uptake (% ID/g) for ⁹⁰Y-MOv18 was 53.7 and 7.4 in A431FR and A431tMock respectively; corresponding values for ¹¹¹In-Mov18 were 45.0 and 11.3. These data demonstrate the feasibility of ⁹⁰Y-labeling of MOv18 without compromising antibody binding ability and the immunoreagent-specific localization in vivo on FR-expressing tumors, suggesting the suitability of ⁹⁰Y-MOv18 for clinical studies.Angela Coliva and Alberto Zacchetti contributed equally to this work.     

Localization of an anti-CEA monoclonal antibody in colo-rectal carcinoma xenografts

Summary A mouse monoclonal anti-CEA antibody (11.285.14) has been examined for tumour localization potential by assessing its distribution in immunodeprived mice with xenografts of human colon carcinoma cell lines HCT-8, HRT-18, HT-29, and LS174T and a xenograft (HRVB) established from a primary rectal carcinoma. With four carcinomas (HCT-8, HT-29, LS174T, and HRVB) preferential tumour localization of ¹²⁵I anti-CEA was seen. Compared with ¹³¹I normal IgG₁ localization indices of up to 4.4:1 were achieved. Up to 10% of the injected dose of ¹²⁵I anti-CEA was present/g of tumour tissue and with the largest xenografts examined (3–4 g) up to 40% of the total body reactivity was localized in tumour tissue. The tumour localization of ¹³¹I labelled antibody was visualized by external gamma camera imaging. Overall antibody localization correlated with the CEA content of the xenografts and the fourth colon carcinoma xenograft (HRT-18) and an osteogenic sarcoma xenograft (791T), both with very low CEA levels, showed no localization of the monoclonal antibody.     

Characterization of a mouse/human chimeric monoclonal antibody (17-1A) to a colon cancer tumor-associated antigen

Mouse monoclonal antibody 17-1A is specific for an antigen expressed on cells of human gastrointestinal malignancies and has been used in radioimmune imaging and therapy trials for patients with colon and pancreatic cancer. The cell line SG3/5 was generated by transfection of a nonproducing mouse myeloma line (SP2/0) with a chimeric gene construct composed of variable regions from the mouse 17-1A immunoglobulin (gamma 2a, kappa) and constant regions of human k and gamma 3 immunoglobulin genes. The secreted immunoglobulin was bound by mouse monoclonal antibodies to human IgG(Fc) and IgG3 but not by staphylococcal protein A. Gel filtration HPLC profiles of purified chimeric antibody were similar to normal human IgG3 but quite different from native 17-1A and normal human IgG1, 2, and 4. Native and chimeric 17-1A had similar patterns of reactivity with colon cancer, other adenocarcinoma, and leukemic cell lines. Competitive inhibition documented that native and chimeric 17-1A had identical capacities to inhibit radiolabeled native 17-1A binding to colon cancer cell lines. Thus, the chimeric 17-1A exhibits molecular characteristics of normal human IgG3 but retains the specificity and binding affinity of the native 17-1A murine monoclonal antibody. The native and chimeric 17-1A mediated similar modest degrees of human lymphocyte and monocyte ADCC in a 4-hr 51Cr release assay, and both failed to mediate complement lysis of colon carcinoma cell lines in the presence of human complement. This human/mouse chimeric monoclonal antibody may be a good candidate for use in clinical trials because it retains the tumor antigen specificity and human effector cell recognition of the native 17-1A, would presumably have a fivefold to 10-fold longer circulating half-life in man, and should be considerably less immunogenic as compared with native murine immunoglobulins.     

Separation and concentration of Δ-6-keto-PGF1α using monoclonal antibody to ω3-olefin structure of trienoic prostanoids

A monoclonal antibody against cis-3-hexen-1-ol was prepared and used to separate and/or concentrate Δ¹⁷-6-keto-prostaglandin F_1α (PGF_1α) in the human sera. cis-3-Hexen-1-ol was conjugated with the human serum albumin (HSA) according to the N-succinimidylester method and hyperimmunized to BALB/c mouse. The monoclonal afntibodies were obtained from hybridoma clones established by a fusion between SP2/0-Ag14-k13 mouse myeloma cells and splenocytes of a mouse. A monoclonal antibody, named 4G9-12B, recognized the epitope characteristic for ω3-olefin structure. The 4G9-12B antibody became more specific for Δ¹⁷-6-keto-PGF_1α than 6-keto-PGF_1α by applying inhibition ELISA using amino-residue coating plates. Using the prepared immunoaffinity columns of this antibody, Δ¹⁷-6-keto-PGF_1α was clearly detected in 6 pg/ml of the human blood sera by GC/MS analysis. These results suggest that the monoclonal antibody to the partial structure of trienoic prostanoid, ω3-olefin unit, and that its immunoaffinity columns are useful in separating and concentrating Δ¹⁷-6-keto-PGF_1α in the human blood or urine.     

Radioactive gold cluster immunoconjugates: Potential agents for cancer therapy

An 11 gold atom (undecagold) cluster was covalently attached to specific sites on Fab′, F(ab′)₂ and whole IgG molecules such that each carried 11–33 gold atoms without significant loss of native immunospecificity. Gold cluster labeled 17-1A monoclonal F(ab′)₂ antibody fragments showed 80% immunoreactivity compared to native antibody fragments in binding to human colon carcinoma cells in vitro. Radioactive gold in vivo biodistributions in nude mice with human tumors are also reported. By using clusters, potentially a larger destructive payload can be carried per antibody.     

An optimized antibody-chelator conjugate for imaging of carcinoembryonic antigen with indium-111

A monoclonal antibody to carcinoembryonic antigen showing minimal cross-reactivity with blood cells and normal tissues was derivatized with benzylisothiocyanate derivatives of EDTA and DTPA. Seven chelators per immunoglobulin could be incorporated without loss of immunoreactivity. The resulting conjugates, labeled with indium-111, showed low liver uptake in animals. A cold kit, comprising the DTPA conjugate at a molarity of antibody bound chelator exceeding 1 x 10⁻⁴M, gave radiochemical yields of indium labeled antibody of ⩾95% and was stable for 1 yr.     

Clinical studies with B72.3 in ovarian cancer and probe-guided tumor localization

The present study reports the findings of radiolabeled monoclonal antibody (MoAb) application in the post-operative monitoring of patients with ovarian carcinoma. We studied 40 patients with histologically confirmed ovarian cancer using two radiolabeled MoAbs, ¹³¹I-B72.3 and ¹³¹I-OC125. Antibody scans showed that high uptake of radiolabeled MoAb was consistently associated with presence of disease confirmed by computed tomography, ultrasonography, clinical evolution and in a few cases by second look procedures. Computed tomography could not discriminate well between post-operative fibrosis and tumor lesion especially when localized in the peritoneum. We conclude that radiolabeled MoAbs, computed tomography and serum tumor markers are complementary in the postoperative monitoring of patients with ovarian carcinoma.