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PubMedDate not supplied

Thermally stabilized immunoconjugates: conjugation of antibodies to alkaline phosphatase stabilized with polymeric cross-linkers.

A method of conjugating poly(glutamic acid) poly(phosphorothioate)-cross-linked alkaline phosphatase to maleimide-derivatized immunoglobulin is described. Intramolecular autocatalyzed cross-linking of alkaline phosphatase at 2:1 to 4:1 polymer:enzyme ratios introduced 32-68 thiolates on the surface of the enzyme. Depending on the stoichiometry of polymer to enzyme, the cross-linked alkaline phosphatase retained 75-90% of its native catalytic activity. The cross-linked thiolate-functionalized alkaline phosphatase was conjugated to maleimide-derivatized immunoglobulin. Compared to a control prepared using non-cross-linked alkaline phosphatase, these conjugates were smaller in size and more stable to heat. The enzymatic activity of the cross-linked conjugates after incubation at 45 degrees C and pH 7.5 for 25 days was 35% higher than those of the highest-activity control conjugates. The conjugation process could be controlled by varying the stoichiometries of poly(glutamic acid) poly(phosphorothioate), alkaline phosphatase, and immunoglobulin.

Alkaline Phosphatase↗
PubMed1995

Preparation and preclinical evaluation of humanised A33 immunoconjugates for radioimmunotherapy.

A humanised IgG1/k version of A33 (hA33) has been constructed and expressed with yields up to 700 mg l-1 in mouse myeloma NS0 cells in suspension culture. The equilibrium dissociation constant of hA33 (KD = 1.3 nM) was shown to be equivalent to that of the murine antibody in a cell-binding assay. hA33 labelled with yttrium-90 using the macrocyclic chelator 12N4 (DOTA) was shown to localise very effectively to human colon tumour xenografts in nude mice, with tumour levels increasing as blood concentration fell up to 144 h. A Fab' variant of hA33 with a single hinge thiol group to facilitate chemical cross-linking has also been constructed and expressed with yields of 500 mg l-1. Trimaleimide cross-linkers have been used to produce a trivalent Fab fragment (hA33 TFM) that binds antigen on tumour cells with greater avidity than hA33 IgG. Cross-linkers incorporating 12N4 or 9N3 macrocycles have been used to produce hA33 TFM labelled stably and site specifically with yttrium-90 or indium-111 respectively. These molecules have been used to demonstrate that hA33 TFM is cleared more rapidly than hA33 IgG from the circulation of animals but does not lead to accumulation of these metallic radionuclides in the kidney. 90Y-labelled hA33 TFM therefore appears to be the optimal form of the antibody for radioimmunotherapy of colorectal carcinoma.

Amino Acid Sequence↗
PubMed1999

Targeted alpha therapy: evidence for potential efficacy of alpha-immunoconjugates in the management of micrometastatic cancer.

There can be little doubt that one of the most important problems in the management of cancer is control of metastatic disease. This objective must be achieved ideally with a systemic therapeutic modality that targets cancer cells and gives minimal collateral damage to critical normal cells. The efficacy of targeted cancer therapy relies on the ability of a toxin to be located in the target cancer cell. The ideal toxin is one that is active only in the cancer cell, and not in critical normal cells. Failing this, the next best approach is a toxin with a short effective lifetime to target early stage micrometastatic disease. This rules out chemical toxins, given that they remain effective until excreted from the body, and localization of dose to the cancer cell rules out beta-emitting radio-isotopes (RI). Alpha-emitting RI, however, are much more appropriate toxins because they are short-lived and because their cytotoxicity is the result of their high rate of energy loss and short range of the alpha particles. These radionuclides have properties that are particularly suited for the elimination of single cells in transit or small nests of cancer cells. In vitro and in vivo experiments with alpha RI show dramatic superiority over beta RI. Only a few nuclear hits are needed to kill cells, and the formation of metastatic lung lesions and subcutaneous lesions in mice can be inhibited by systemic administration of alpha emitters. But alpha RI have not been able to control solid tumours, for which beta RI are better suited. A small number of alpha-emitting radionuclides are currently under investigation. These are terbium (Tb)-149, astatine (At)-211, bismuth (Bi)-212 and Bi-213. Terbium-149 and At-211 both require accelerators in close proximity to the place of application. The Bi isotopes are produced by long-lived parents and, as such, can be obtained from generators. The first phase-1 dose escalation trial with Bi-213 radioimmunoconjugate (RIC) commenced in New York in 1997, and other trials are planned with At-211 RIC and At-211 methylene blue for melanoma. Actinium (Ac)-225 is obtained from the decay of thorium (Th)-229, which is a waste product in the enrichment of fissile Th-233. Alternative accelerator production routes are being investigated, beginning with the European Centre for Nuclear Research (CERN) GeV proton spallation source. The ready and low-cost availability of the Ac:Bi generator is an important element in the implementation of clinical trials for patients with poor prognoses but without evidence of metastatic disease.

Alpha Particles↗
PubMed2000

Targeted photodestruction of human colon cancer cells using charged 17.1A chlorin e6 immunoconjugates.

The goal of this study was to develop a strategy for the selective destruction of colorectal cancer cells. Towards this end, photoimmunoconjugates were prepared between the anti-colon cancer monoclonal antibody 17.1A and the photosensitizer (PS) chlorin(e6) (c(e6)). Polylysine linkers bearing several c(e6) molecules were covalently attached in a site-specific manner to partially reduced IgG molecules, which allowed photoimmunoconjugates to bear either cationic or anionic charges. The conjugates retained immunoreactivity as shown by enzyme-linked immunosorbent assays and by competition studies with native antibody. The overall charge on the photoimmunoconjugate was an important determinant of PS delivery. The cationic photoimmunoconjugate delivered 4 times more c(e6) to the cells than the anionic photoimmunoconjugate, and both 17.1A conjugates showed, in comparison to non-specific rabbit IgG conjugates, selectivity for antigen-positive target cells. Illumination with only 3 J cm(-2) of 666 nm light reduced the number of colony forming cells by more than 90% for the cationic 17.1A conjugate and by 73% for the anionic 17.1A conjugate after incubation with 1 microM c(e6) equivalent of the respective conjugates. By contrast, 1 microM free c(e6) gave only a 35% reduction in colonies. These data suggest photoimmunoconjugates may have applications in photoimmunotherapy where destruction of colorectal cancer cells is required.

Antibodies, Monoclonal↗
PubMed2000

Clinical studies of new "biologic" approaches to therapy of acute myeloid leukemia with monoclonal antibodies and immunoconjugates.

Conventional chemotherapeutic treatments of acute leukemias are often associated with life-threatening toxic effects due to a lack of specificity for hematopoietic cells. Monoclonal antibodies and fusion proteins that target antigens on leukemic blasts are being explored for their antileukemic effects and as a means of delivering chemotherapy or radiation directly to malignant cells. This approach might be safer and more effective than current non-specific chemotherapeutic agents. The cell surface antigens CD33 and CD45 are attractive targets. Although CD33 is expressed on acute myelocytic leukemic blast cells from about 90% of patients, normal hematopoietic stem cells lack this antigen, as do essentially all non-hematopoietic tissues. Anti-CD33 antibodies have been engineered to selectively target malignant myeloid and immature normal cells while sparing normal stem cells. Recently, anti-CD33 antibodies have also been used to deliver radiation or a cytotoxic agent directly to leukemic cells. The strategy for using CD45 as a target differs from CD33 in that it is expressed not only by the vast majority of leukemias, but also by normal stem cells. Therefore, 131I-labeled anti-CD45 antibody has been used in combination with conventional preparative regimens for patients receiving marrow transplantation for acute leukemia. Because the receptor for granulocyte-macrophage colony-stimulating factor is expressed by most myeloid leukemias, fusion proteins consisting of granulocyte-macrophage colony-stimulating factor ligand associated with diphtheria toxin have been proposed as a means of delivering a toxic agent directly to leukemic cells. Both unconjugated and conjugated antibodies show significant promise in the treatment of acute myelocytic leukemia.

Antibodies, Monoclonal↗
PubMed2002

A phase I study of 99mTc-hR3 (DiaCIM), a humanized immunoconjugate directed towards the epidermal growth factor receptor.

A phase I trial was conducted to evaluate the safety, tumour and normal tissue localization, pharmacokinetics and radiation dosimetry of Tc-hR3, a humanized monoclonal antibody directed towards the epidermal growth factor receptor, in 12 patients with recurrent or metastatic epithelial malignancies. Patients were injected intravenously with 3.0 mg or 6.0 mg (1010 MBq) of Tc-hR3. Blood and plasma concentrations of radioactivity were measured and a complete 24 h urine collection was obtained. Whole-body images were acquired up to 24 h post-injection and normal organ uptake quantified. Radiation dosimetry was estimated using MIRDose. Safety was evaluated by clinical observation, biochemical/haematological testing and by measuring immune response to Tc-hR3. There were no adverse effects, no changes in biochemical/haematological indices and no immune response to Tc-hR3. Tc-hR3 was rapidly cleared from the blood with a distribution half-life of 10.8+/-3.8 min. The volume of distribution, and clearance, were 180+/-37 ml.kg and 14+/-3 ml.kg.min, respectively. The elimination phase could not be discerned due to increasing blood radioactivity at later times. About 19-24% was excreted in the urine. Normal tissue uptake was mainly in the liver (44-50%), spleen (3-4%) and kidneys (3%). Imaging was positive in one patient with squamous cell carcinoma of the mouth and an involved cervical lymph node. The whole-body radiation dose from Tc-hR3 was 1.34+/-0.02x10 mSv.Bq. We conclude that Tc-hR3 exhibited an excellent safety profile. Future studies to determine the sensitivity and specificity of imaging with Tc-hR3 in a larger group of patients with pre-selection for epidermal growth factor receptor positivity are planned.

Adolescent↗
PubMed1998

Immunobiology of lipopolysaccharide (LPS) and LPS-derived immunoconjugates vaccinate mice against Salmonella typhimurium.

The immunobiology of lipopolysaccharide (LPS) of Salmonella typhimurium LT2-71 was studied in its native, modified and conjugated states using mice as the experimental model. An alkali-treated detoxified fraction of LPS (D-LPS) was found to be not only non-toxic but also equally immunogenic, like LPS. In addition D-LPS alone or conjugated with enterotoxin or hemolysin was also non-pyrogenic and non-indurogenic. The immunoprophylactic activity of D-LPS conjugates to a 100 ID50 challenge dose of S. typhimurium was also higher than that of detoxified LPS or native LPS.

Animals↗
PubMed1995

Intratumoral injection of an adriamycin immunoconjugate against human pancreatic cancer xenografts.

We have evaluated the effect of an adriamycin conjugate of monoclonal antibody Nd2 (ADM-Nd2) on the growth rate of SW1990 xenografts grown subcutaneously in athymic nude mice. Intravenous or intraperitoneal administration of radiolabeled Nd2 resulted in a maximum tumor accumulation of approximately 45% of the initial dose/g of tumor 3-7 days after administration. However, administration into the tumor produced retention of 1200%ID/g 1 day after, with 50% of this high value remaining even at 7 days after administration. In contrast, intratumoral administration of a non-specific immunoglobulin showed a lower initial retention and rapid loss of label. Both intravenously and intratumorally administered ADM-Nd2 reduced the growth rate of SW1990 xenografts. While a single intravenous administration arrested growth for about two weeks, a single intratumoral injection prevented any increase in tumor size even 45 days after administration. Xenografts treated with ADM-Nd2 showed degenerative changes at the histological level. Neither Nd2 alone nor Adriamycin alone inhibited growth when administered at the same dose as the conjugate.

Animals↗
PubMed2003

Pharmacokinetics and biodistribution of the antitumor immunoconjugate, cantuzumab mertansine (huC242-DM1), and its two components in mice.

The humanized monoclonal antibody maytansinoid conjugate, cantuzumab mertansine (huC242-DM1) that contains on average three to four linked drug molecules per antibody molecule was evaluated in CD-1 mice for its pharmacokinetic behavior and tissue distribution, and the results were compared with those of the free antibody huC242. The pharmacokinetics in blood were similar for (125)I-labeled conjugate and antibody with terminal half-lives of 154 and 156 h, respectively. Pharmacokinetic analysis using an enzyme-linked immunosorbent assay (ELISA) method, which measures intact conjugate in plasma samples revealed a faster clearance for the conjugate corresponding to a half-life of 42.2 h. This faster clearance is explained as the result of clearance from circulation and concomitant clearance of drug from circulating conjugate through linker cleavage. An antibody-specific ELISA allowed the determination of the clearance rate of the antibody component from circulation. The drug clearance rate from circulating conjugate was then calculated as the difference between the clearance of the conjugate and the clearance of the antibody component and found to be about three times that of the antibody component. The above results were confirmed with a conjugate, huC242-[(3)H]DM1, where the linked DM1 drugs carried a stable tritium label. Tissue distribution studies with (125)I-labeled conjugate and antibody showed antibody-like behavior for the conjugate; the antibody of the conjugate did not distribute or bind significantly to any solid tissue.

Animals↗
PubMed2004

Improved targeting of photosensitizers by intratumoral administration of immunoconjugates.

Biodistribution of technetium (99mTc) labeled hematoporphyrin derivative (HpD, Photosan-3) conjugated to a monoclonal antibody to carcinoembryonic antigen (anti-CEA) was compared following intravenous (i.v.) and intratumoral (i.t.) administration in solid Ehrlich ascites tumor bearing mice. Images of mice at different time intervals were acquired after injection of radiolabeled PS-3 in either conjugated or unconjugated forms. Quantitative estimation of the radiolabel in different tissues was performed by selecting the different region of interests (ROIs). Maximum accumulation of both free and antibody conjugated PS-3 following i.v. administration was observed in liver followed by tumor. Tumor/muscle (T/N) ratio was more with free PS-3 compared to conjugated PS-3. Pharmacokinetics of free and conjugated PS-3 was also different with faster accumulation of conjugated PS-3 in the tumor. With intratumoral administration of anti-CEA-PS-3-99mTc, specific accumulation and retention of the sensitizer was observed in the tumor tissue. Since, direct injection of antibody conjugated photosensitizer into the tumor resulted in longer retention of the dye in the tumor with no accumulation in the normal tissues, the present results imply that the toxicity to normal tissues could be reduced significantly with selective destruction of the tumor following photodynamic treatment with the use of i.t. administration of specific antibodies conjugated to photosensitizers.

Animals↗
PubMed2000

Influence of immunogenicity on the pharmacokinetics of BMS-191352, a Pseudomonas exotoxin immunoconjugate, in rats and dogs.

BMS-191352 is an immunotoxin construct of modified Pseudomonas exotoxin conjugated to a fragment of the BR96 monoclonal antibody. We have investigated the potential for immunogenicity of BMS-191352 and its influence on the pharmacokinetics in rats and dogs. BMS-191352 was administered intravenously at doses of 0.75, 1.5, and 3 mg m(-2) once every two days for a total of five doses in rats, and 1.2, 2.4, and 4.8 mg m(-2) once every three days for a total of five doses in dogs. Blood samples were collected on days 1 and 9 in rats, and on days 1, 7, and 13 in dogs to monitor pharmacokinetics and anti-BMS-191352 immune response. Plasma concentrations of BMS-191352 and serum anti-BMS-191352 antibody titre were determined using ELISA assays. Pharmacokinetics were assessed using a non-compartmental method. Anti-BMS-191352 antibodies were not observed in rats within the drug administration interval. In all dogs, except one, markedly higher anti-BMS-191352 antibody titres were observed on day 13 compared with days 1 and 7, and its magnitude was independent of BMS-191352 dose. The single dose kinetics of BMS-191352 in rats and dogs were linear and the drug exposures were generally dose proportional. Mean half-life, total body clearance, and volume of distribution were 1.74 h, 3.35 mL min(-1) m(-2), and 0.27 Lm(-2) in rats, respectively, and 4.27 h, 6.28 mL min(-1) m(-2), 1.19 L m(-2) in dogs, respectively. The multiple-dose (day 9) kinetics in rats were similar to the single-dose kinetics. In dogs, the disposition of BMS-191352 on day 7 was similar to that on day 1; however, there was a precipitous reduction in the systemic drug exposure (by 5- to 110-fold) and marked increase in drug clearance on day 13. These changes in the kinetics of BMS-191352 were attributed to the generation of anti-BMS-191352 antibodies. In the one dog that did not develop anti-BMS-191352 antibodies, the pharmacokinetics were unchanged. The pharmacokinetics of BMS-191352 may be perturbed due to an immune response thus restricting the therapeutic utility of the immunotoxin.

ADP Ribose Transferases↗
PubMed2005

Synthesis and biological evaluation of a paclitaxel immunoconjugate.

Targeted cancer therapy is a promising strategy for the treatment of this disease. In this approach, a cytotoxic agent (CA), such as a drug or a radionuclide, is attached, usually covalently, to a "targeting" vehicle (TV), which in turn is capable of recognizing specific receptor motifs on the surface of the tumor cells. Once administered systemically, the construct would localize on the tumor through the TV moiety and would release the CA cargo, resulting in the destruction of the malignant tissue. Small-molecule peptides as well as monoclonal antibodies have been used as TVs. The synthesis, antigen binding, and cytotoxicity of a covalent conjugate of the anticancer drug paclitaxel (taxol) to the anti-epidermal growth factor receptor (EGFR) monoclonal antibody C225 (IMC-C225; Erbitux, ImClone Systems, Somerville, NJ) are described in this chapter to illustrate the methods used for the construction and in vitro evaluation of these conjugates.

Antibodies, Monoclonal↗
PubMed2002

Selective intracellular delivery of dexamethasone into activated endothelial cells using an E-selectin-directed immunoconjugate.

In chronic inflammatory diseases, the endothelium is an attractive target for pharmacological intervention because it plays an important role in leukocyte recruitment. Hence, inhibition of endothelial cell activation and consequent leukocyte infiltration may improve therapeutic outcome in these diseases. We report on a drug targeting strategy for the selective delivery of the anti-inflammatory drug dexamethasone to activated endothelial cells, using an E-selectin-directed drug-Ab conjugate. Dexamethasone was covalently attached to an anti-E-selectin Ab, resulting in the so-called dexamethasone-anti-E-selectin conjugate. Binding of the conjugate to E-selectin was studied using surface plasmon resonance and immunohistochemistry. Furthermore, internalization of the conjugate was studied using confocal laser scanning microscopy and immuno-transmission electron microscopy. It was demonstrated that the dexamethasone-anti-E-selectin conjugate, like the unmodified anti-E-selectin Ab, selectively bound to TNF-alpha-stimulated endothelial cells and not to resting endothelial cells. After binding, the conjugate was internalized and routed to multivesicular bodies, which is a lysosome-related cellular compartment. After intracellular degradation, pharmacologically active dexamethasone was released, as shown in endothelial cells that were transfected with a glucocorticoid-responsive reporter gene. Furthermore, intracellularly delivered dexamethasone was able to down-regulate the proinflammatory gene IL-8. In conclusion, this study demonstrates the possibility to selectively deliver the anti-inflammatory drug dexamethasone into activated endothelial cells, using an anti-E-selectin Ab as a carrier molecule.

Antibodies↗
PubMed2000

Experimental photoimmunotherapy of hepatic metastases of colorectal cancer with a 17.1A chlorin(e6) immunoconjugate.

Photoimmunotherapy (using a monoclonal antibody-targeted photosensitizer and red light) may be a strategy to overcome the limitations inherent in photodynamic therapy of liver tumors. The aims of this study were (a) to test the efficacy of selective treatment of hepatic metastases of colorectal cancer in an orthotopic murine xenograft using the murine monoclonal antibody 17.1A conjugated to the photosensitizer chlorin(e6), and (b) to compare the tumor response after the same light dose was delivered at two different fluence rates. Based on previous biodistribution studies that had shown that the photoimmunoconjugate with a polyanionic charge had both a higher absolute tumor chlorin(e6) content and a greater tumor:normal liver ratio than those obtained with a photoimmunoconjugate bearing a polycationic charge, mice were treated 3 h after i.v. injection of the polyanionic 17.1A chlorin(e6) conjugate or unconjugated photosensitizer. Red light was delivered into the liver tumor by an interstitial fiber, and tumor response end points were total tumor weight in the short term and survival in the long term. There was a highly significant reduction (<20% of controls; P = 0.0035) in the weight of the tumors in the mice treated with photoimmunotherapy, and the median survival increased from 62.5 to 102 days (P = 0.015). Photodynamic therapy with free chlorin(e6) produced a smaller decrease in tumor weight and a smaller extension of survival, neither of which were statistically significant. A comparison of photoimmunotherapy with 10 J of light delivered at 30 or 300 mW showed that the higher fluence rate prolonged survival significantly more than the lower fluence rate. This may have been because the high fluence rate gave a contribution of laser-induced hyperthermia to the photodamage. Correlation studies showed that the amount of normal liver remaining at necropsy correlated best with survival. Photoimmunotherapy shows efficacy in destroying liver tumors, and future studies should maximize selectivity to minimize the destruction of normal liver.

Animals↗
PubMed2003

Targeting colon cancer cells with genistein-17.1A immunoconjugate.

We have shown that genistein, a major component of soy, has anti-colon cancer effects in vitro. These effects are attainable at high concentrations that are difficult to achieve in the serum. The purpose of this study was to enhance the activity of genistein against colon cancer cells by coupling it to 17.1A. The monoclonal antibody 17.1A recognizes an epithelial membrane antigen that is overexpressed in colon cancer. Synthesis of Gen-17.1A was achieved by photochemical conjugation using sulfa-SANPAH. Its purity was evaluated by SDS-PAGE. Binding of Gen-17.1A to SW-620 and HT-29 cells was shown using flow cytometry. Internalization was demonstrated by FITC-labeling. Gen-17.1A induced apoptosis in colon cancer cells as evidenced by the acridine orange/ethidium bromide staining method. Gen-17.1A significantly inhibited colon cancer cell growth in vitro and in vivo. Our findings suggest that conjugating genistein to 17.1A monoclonal antibody enhances its effects against colon cancer cells.

Animals↗
PubMed2005

In vivo drug-linker stability of an anti-CD30 dipeptide-linked auristatin immunoconjugate.

Effective antibody-drug conjugates (ADC) combine high drug-linker stability in circulation and efficient intratumoral release of drug. Conjugation of monomethyl auristatin E (MMAE) to the anti-CD30 monoclonal antibody (mAb), cAC10, produced a selective and potent ADC against CD30(+) anaplastic large cell lymphoma and Hodgkin's disease models. This ADC, cAC10-valine-citrulline-MMAE, uses a protease-sensitive dipeptide linker designed to release MMAE by lysosomal cathepsin B in target cells but maintain a stable linkage and attenuate drug potency in circulation. To evaluate ADC stability in vivo, we developed methods for measuring drug/mAb ratios at progressive times in plasma from ADC-treated mice and nonhuman primates. Anti-idiotype mAb permitted the capture and quantitation of mAb cAC10, whereas antidrug mAb and MMAE-conjugated horseradish peroxidase reporter provided quantitative detection of conjugated drug following its in vitro release by cathepsin B. These data were validated by an alternative ELISA using anti-idiotype and anti-MMAE mAbs for capture and detection, respectively. Both methods differentiated ADC with variable levels of drug loading and were subsequently applied to stability studies in severe combined immunodeficient mice and cynomolgus monkeys. Evaluation of ADC from mouse circulation showed the linker half-life to be approximately 144 hours (6.0 days), significantly greater than that reported for disulfide- or hydrazone-linked ADCs in mice or human trials. In cynomolgus monkey, the apparent linker half-life was approximately 230 hours (9.6 days), suggesting that the drug-linker will be highly stable in humans. These data represent the longest reported drug-linker half-life to date and provide the basis for the pronounced specificity and antitumor activity of cAC10-valine-citrulline-MMAE.

Animals↗