Comparative localization of murine monoclonal antibody Me1-14 F(ab')2 fragment and whole IgG2a in human glioma xenografts

Monoclonal antibodies (MAbs) targeted to glioma-associated antigens may allow the selective delivery of imaging and therapeutic agents to brain tumors; the use of MAb fragments may be a strategy to further improve tumor uptake of such agents relative to normal tissues. In this study, we have examined the in vivo localization of radioiodinated MAb Me1-14, a murine immunoglobulin G2a (IgG2a) reactive with gliomas, and its F(ab')2 fragment in s.c. and intracranial xenografts of human glioma cell line D-54 MG in athymic mice. The radiolabeled F(ab')2 fragment of Me1-14 was demonstrated to possess in vitro binding affinity and immunoreactivity comparable to that of whole IgG. Direct comparison of IgG and F(ab')2 biodistribution in s.c. xenograft-bearing mice showed higher tumor: normal tissue ratios of the F(ab')2 fragment compared to IgG. In intracranial tumor-bearing mice paired-label analysis using a nonspecific protein control showed earlier specific tumor localization by the F(ab')2 fragment of Me1-14 compared to IgG. Blood-to-tumor transfer constants (K1) derived for Me1-14 F(ab')2 were significantly greater than those for whole Me1-14 (P = 0.01). Estimated radiation dosimetry revealed that 131I-labeled Me1-14 F(ab')2 would deliver higher radiation doses to tumor than to normal tissues. These studies demonstrate that the F(ab')2 fragment of Me1-14 may be a potential agent for immune-directed brain tumor diagnosis and therapy.     

Selective enhancement of antitumor activity of N-acetyl melphalan upon conjugation to monoclonal antibodies

Melphalan (MEL) is an aromatic alkylating agent which is useful for the treatment of a number of human cancers, including myeloma and ovarian cancer. However, like most cytotoxic drugs, MEL has side effects, due to its nonspecific effects on all or most cells. To overcome these nonspecific effects N-acetyl melphalan (N-AcMEL) was synthesized and found to be 75 times less toxic to tumor cells in vitro. However, when N-AcMEL was conjugated to monoclonal antibodies (MoAbs) to form N-AcMEL-MoAb conjugates the cytotoxic effect of MEL was restored, but with a difference in that the MEL could only act on cells which bound antibody. It was shown that, for N-AcMEL-MoAb conjugates, the N-AcMEL entered cells via the MoAb, by endocytosis, and not by the phenylalanine amino acid transport system. In addition, N-AcMEL-MoAb conjugates more effectively erradicated tumors in vivo than does free MEL or N-AcMEL. The N-AcMEL-MoAb conjugates therefore have high specific activity both in vitro and in vivo and a markedly reduced nonspecific toxicity, as N-AcMEL is relatively nontoxic to cells unless conveyed there by MoAb. In these respects the study offers a new approach to the use of chemotherapeutic agents in patients with cancer.     

Nature of linkage and mode of action of methotrexate conjugated with antitumor antibodies: implications for future preparation of conjugates

The binding of methotrexate (MTX) to IgG in conjugates was examined by studies on a direct MTX conjugate with a monoclonal antibody (aMM46) to mouse mammary tumor MM46 cells and corresponding irrelevant antibody and normal gamma-globulin conjugates, all prepared by the active ester method with MTX N-succinimidyl ester (MTX-OSu). The binding was examined in terms of effects on the potency and selectivity of the cytotoxic activity of the aMM46 conjugate. The results obtained supported the following conclusions: (a) MTX-OSu reacts not only with the amino group of IgG to give an amide bond, but also with another group(s) to give a less stable bond(s) such as an ester bond; (b) in contrast to the amide bond-linked MTX, which is taken up by the cells by endocytic internalization, a substantial portion of the MTX linked by an ester or other less stable bond(s) is released from the conjugates extracellularly and enters the cells by the MTX active transport system, as revealed by the inhibitory effect of thiamine pyrophosphate on the cytotoxicity; (c) this MTX linked by a less stable bond(s) that causes nonspecific cytotoxicity can be removed by treatment with hydroxylamine; (d) the overall cytotoxicity of aMM46-MTX decreased on removal of this less stably linked MTX, suggesting that the lysosomal degradation of the conjugate carrying amide bond-linked MTX to liberate MTX derivatives of low molecular weight is insufficient; (e) the liberation of low-molecular-weight substances in the lysosomes is probably more important for efficient entry of active substances into the cytosol, than for inhibition of the activity of dihydrofolate reductase, because after hydroxylamine treatment, the amide bond-linked MTX showed greater decrease in drug cytotoxicity than in inhibitory activity against dihydrofolate reductase; (f) in combination with hydroxylamine treatment, insertion between MTX and IgG of a linkage capable of ready cleavage in lysosomes deserves exploitation as a method for making potent conjugates with less nonspecific activity.     

Induction of lasting complete regression of preformed distinct solid tumors by targeting the tumor vasculature using two new anti-endoglin monoclonal antibodies.

Endoglin (EDG, CD105) is a proliferation-associated antigen on endothelial cells. In this study, two new anti-EDG monoclonal antibodies (mAbs) Y4-2F1 (or termed SN6j) and P3-2G8 (SN6k) were generated and used for treating distinct preformed tumors. These mAbs, both IgG1-kappa antibodies, cross-reacted weakly with mouse endothelial cells but defined epitopes different from the epitope defined by a previously reported anti-EDG mAb K4-2C10 (B. K. Seon et al., Clin. Cancer Res., 3: 1031-1044, 1997). SN6j and SN6k reacted strongly with human endothelial cells and vascular endothelium of malignant human tissues but showed no significant reactivity with tumor cells per se. The deglycosylated ricin A chain (dgRA) conjugates of the two mAbs showed a weak but specific cytotoxic activity against murine endothelial cells in vitro. In the therapeutic studies, severe combined immunodeficient mice were inoculated s.c. with MCF-7 human breast cancer cells and left untreated until palpable tumors of distinct size (4-6 mm in diameter) appeared. Mice with the distinct tumors were treated by i.v. administration of individual anti-EDG conjugates, unconjugated mAbs, or a control conjugate. Long-lasting complete regression of the tumors was induced in the majority of tumor-bearing mice (n = 8 for each conjugate) when 40 microg of the individual conjugates were administered three times via the tail vein. It is remarkable that the tumors remained regressed without further therapy for as long as the mice were followed (i.e., 100 days). Control conjugate did not induce regression of the tumors in any of the treated mice, although weak nonspecific effects were observed in some of the mice (n = 8). The effects of unconjugated mAbs were small with the dose used, i.e., 34 microg three times. The anti-EDG conjugates showed antiangiogenic activity in the dorsal air sac assay in mice. The results suggest good potential of these conjugates for the clinical application.     

Sensitive detection of human IgG in ELISA using a monoclonal anti-IgG-peroxidase conjugate

Enzyme-antibody (Ab) conjugates specific for IgG are widely used in indirect immunological assays and have been until recently routinely prepared with polyclonal IgG-specific animal Abs. The use of monoclonal Abs (MAbs) could permit a better standardization of the IgG-specific conjugate reagents but is expected to result in lower reactivity due to the recognition of a single epitope by the MAbs. In this work, we have characterized a monoclonal anti-human IgG-peroxidase (HRP) reagent and compared its reactivity with commercial reagents. The murine C5-1 anti-human IgG MAb was selected for conjugation because of its high affinity (K(a) = 1.9 x 10(10)M), pan-IgG reactivity and absence of cross-reactivity with various structures including animal IgGs. The specific activity and binding kinetics of the C5-1:HRP conjugate were similar to the ones of two polyclonal anti-IgG:HRP conjugates when tested with immobilized human IgG. The C5-1:HRP conjugate could detect low amounts of human IgG much more effectively than two commercial monoclonal conjugates although it was slightly less effective than a polyclonal conjugate. However, the C5-1 conjugate yielded reduced background reactivity compared to the polyclonal conjugate, resulting in similar signal-to-noise ratios. These results indicate that the C5-1:HRP conjugate could be a suitable substitute for anti-human IgG conjugates prepared from animal antisera.     

Effect of tumor size on monoclonal antibody uptake in a metastatic model.

We studied the effect of tumor size on monoclonal antibody (mAb) in a murine hepatic model. Intrasplenic injection of HT-29 LMM metastatic human colon cancer cell line reproducibility results in hepatic metastases formation in congenitally athymic mice. HT-29-15, a murine mAb of the IgG1 class reactive with the HT-29 LMM cell line, and BL-3, an isotype-matched control mAb, were labeled with iodine 125. Labeled mAbs were injected intravenously into mice with hepatic metastases. Animals were sacrificed on day 5, and tumor and normal tissue weighed and counted. Specific mAb uptake (percent injected dose per gram, %ID/g) by hepatic metastases (5.16 +/- 3.96) was significantly greater than nonspecific uptake (1.41 +/- 0.42) (P less than 0.001). %ID/g of tumor was dependent upon the mAb used, and was independent of tumor weight; consequently, a linear correlation between tumor weight and total uptake (%ID) for specific mAb (r = 0.88, P less than 0.0001) and nonspecific mAb (r = 0.99, P less than 0.0001) administration was demonstrated. We have shown both specific and nonspecific mAb uptake per gram of tumor to be a constant for the given mAb/tumor system and with uptake of mAb to be linearly related to tumor weight.     

Clinical application of monoclonal antibody-drug conjugates for immunotargeting chemotherapy of colorectal carcinoma

Monoclonal antibody-drug conjugates were applied as a clinical trial for patients who, based on the experimental study, had colorectal cancer. Monoclonal antibody A7, from a mouse splenocyte immunized against human colon cancer, was used as a drug carrier for colon cancer. The anti-cancer drugs mitomycin C (MMC) and neocarzinostatin (NCS) were bound covalently to A7 to form the conjugates A7-MMC and A7-NCS. The in vitro cytotoxic effects of the conjugates on SW1116 cells were stronger than those on free MMC or NCS. The conjugate A7-NCS, when administered to nude mice, brought about the highest NCS tumor concentration, whereas normal immunoglobulin G (IgG)-NCS distributed evenly in all tissues. The conjugates showed a strong antitumor effect on colon cancer transplanted into nude mice. Forty-one patients with colorectal cancer, including ten patients with postoperative metastasis, were given A7-NCS. The immunoperoxidase and drug concentration studies of the resected specimens showed that NCS was localized specifically in cancer. Patients receiving the conjugate did not experience serious adverse effects. Of the eight patients with postoperative liver metastasis, three showed evidence of tumor reduction on computed tomography (CT) scan and three claimed pain relief. The conjugate did not benefit patients with multiple lung metastasis or peritoneal metastasis.     

The in vitro and in vivo anti-tumour activity of N-AcMEL-(Fab')2 conjugates

To increase the accessibility of drug-antibody complexes to tumours and to decrease non-specific binding via Fc receptors N-acetyl-melphalan (N-AcMEL) was conjugated to F(ab')2 fragments. These fragments were synthesised by pepsin degradation of IgG MoAb. Up to 20 molecules of N-AcMEL could be successfully coupled to each F(ab')2 fragment (compared with 25 molecules/intact IgG) with retention of both drug and antibody activity. The N-AcMEL-F(ab')2 conjugates demonstrated specific cytotoxicity in vitro however despite the absence of non specific Fc receptor binding and greater permeability when using F(ab')2 fragments, the N-AcMEL-F(ab')2 and N-AcMEL-IgG conjugates had similar anti-tumour activity in vivo. Conjugates made with whole IgG and F(ab')2 were equally effective in eradicating subcutaneous solid tumours in mice when injected intravenously. The lower immunogenicity of F(ab')2 fragments compared with whole IgG and the similar cytotoxicity of their conjugates, suggests that the F(ab')2 conjugate has greater clinical utility.     

Interstitial transport of rabbit and sheep antibodies in normal and neoplastic tissues

Interstitial transport of fluorescein isothiocyanate-conjugated nonspecific polyclonal rabbit and sheep IgG was studied in normal (mature granulation) and neoplastic (VX2 carcinoma) tissues grown in a rabbit ear chamber. The interstitial concentration gradients after i.v. injection were analyzed to yield effective interstitial diffusion coefficients, Deff. The one-dimensional diffusion model underestimated Deff by a factor of up to 3 when compared with a two-dimensional model. Despite marked heterogeneities in Deff, the average values of Deff were higher in tumors than in normal tissue. Rabbit IgG moved faster in tumors than the sheep IgG by a factor of 2. When compared with the dextran of same molecular weight, the ratio of Deff between neoplastic and normal tissue decreased from 33 (dextran) to between 2 and 5 (sheep IgG and rabbit IgG, respectively). When compared with dextran of the same Stokes-Einstein radius, IgGs had a lower Deff in both tissue types. These results are consistent with the size, charge, and configuration of antibodies and the structure and charge of the interstitial matrix and suggest that the delivery of antibodies to tumors may be improved by modulating their charge, hydrophilicity, and antigenicity. The molecular weight dependence of Deff also provides a rational basis for the use of bifunctional antibodies and antibody-enzyme conjugates to increase the delivery of low molecular weight anticancer agents to solid tumors.     

Effect of methotrexate-monoclonal anti-prostatic acid phosphatase antibody conjugate on human prostate tumor

Methotrexate (MTX) was conjugated to an immunoglobulin G1 (IgG1) monoclonal antibody specific for human prostatic acid phosphatase (PAP) by the active ester method. The molar ratio of MTX to IgG was 14. MTX-monoclonal antibody conjugate retained substantially the original PAP-binding inhibition activity of the monoclonal antibody. Both MTX-monoclonal antibody conjugate and an identically prepared MTX-normal mouse IgG conjugate preserved 90% of the original dihydrofolate reductase inhibitory activity of MTX. [3H]MTX conjugated to monoclonal anti-PAP antibody was significantly accumulated more in PAP-producing human prostate tumor LNCaP cells than its normal mouse IgG counterpart. No statistical difference was found between the uptake of [3H]MTX conjugated to monoclonal antibody and that of [3H]MTX conjugated to normal mouse IgG by control PAP nonproducing thyroid tumor cells (TT). The antitumor effect of the conjugate was evaluated in vitro by its inhibition on deoxy[6-3H]uridine incorporation into LNCaP cells. The inhibition by MTX-monoclonal antibody conjugate was significantly higher than that by MTX-normal mouse IgG conjugate at 8 micrograms of drug per ml, although it was significantly less than that by free MTX. However, an in vivo tumor and tissue distribution study of [3H]MTX and its conjugates revealed that, 5 days after i.v. administration, [3H]MTX conjugated to monoclonal antibody was preferentially accumulated in LNCaP prostate tumor. Tumor:blood ratios for [3H]MTX, [3H]MTX-monoclonal antibody conjugate, and [3H]MTX-normal mouse IgG conjugate were 1.47, 5.06, and 1.26, respectively. Preliminary results obtained from a pilot study with a small number of animals demonstrated that multiply injected MTX-monoclonal antibody conjugate retarded the growth of xenografted prostate tumor (LNCaP) as compared with the control groups, including free MTX which showed a shorter period of therapeutic effectiveness. This study suggests that MTX conjugated to monoclonal anti-PAP antibody could be a potential reagent for experimental immunochemotherapy of prostate tumor, should the initial in vivo data be extended and confirmed.     

Lymphoscintigraphy of human colorectal carcinoma metastases in athymic mice by use of radioiodinated B72.3 monoclonal antibody

The potential of radioiodinated monoclonal antibody B72.3 for lymphoscintigraphy was evaluated, using suitable animal models of a human colorectal carcinoma. LS174T xenografts were grown at various sites in beta-estradiol-pretreated athymic mice, and the development of metastases in different organs was assessed histologically. After iv inoculation of the mice, 66% of the animals developed "metastases" to the axillary lymph nodes. Of these mice, 100% also developed multiple tumors on their backs and 79% had lung micrometastases. Livers, kidneys, and spleens showed no evidence of tumor growth. In 33% of the mice in which primary LS174T tumors had been removed from the hindfoot pad, metastases to the popliteal lymph nodes were observed 3 1/2 weeks after tumor implantation. BALB/c (nu/nu) female mice bearing axillary and popliteal lymph node metastases were used to test the potential of radiolabeled B72.3 antibody (an IgG1) as a lymphoscintigraphic agent. A monoclonal antibody against horseradish peroxidase (also an IgG1), which did not bind LS174T tumor cells in vitro, served as a control. Both normal and tumor-bearing axillary and popliteal lymph nodes imaged up to 6 hours after the sc injection of 20-40 mu Ci of 125I-labeled B72.3 into either the forefoot or hindfoot pads. The localization index (L.I.) (specific/nonspecific antibody in tumor divided by specific/nonspecific antibody in blood) for LS174T tumors in lymph nodes was approximately 1 during the first 6 hours after antibody injection, thus indicating no specific antibody accumulation. Twenty-four hours and later after sc injection, images of nodal metastases (14-477 mg) and specific antibody accumulations were observed. At these times the L.I.'s ranged 1.5-3.5. Tumor-negative nodes did not image at 24 hours after injection of 125I-labeled B72.3. The L.I.'s of the normal nodes and of other tissues from these mice were about 1.0 at 24 hours, indicating no specific antibody accumulation. Autoradiographic analysis of lymph nodes containing LS174T tumor showed heterogeneous antibody distribution of B72.3 within tumor sections with heavy patches of antibody accumulation in mucin globules. In lymph nodes the normal lymphocytes adjacent to the LS174T tumor cells showed no antibody accumulation. The lack of specific, early antibody accumulation by LS174T tumor-bearing nodes in mice suggests that B72.3 does not accumulate in nodal metastases to the degree necessary to consider it a potential agent for use in lymphoscintigraphy.     

Radiolocalization of xenografted human malignant melanoma by a monoclonal antibody (9.2.27) to a melanoma-associated antigen in nude mice

A murine monoclonal antibody (9.2.27), directed to a Mr 250,000 glycoprotein-chondroitan sulfate proteoglycan complex, was radiolabeled with 125I and assessed for radiolocalization in tumor and normal tissues of normal and tumor-bearing nude mice. The 125I-9.2.27 localized in vivo preferentially in Mr 250,000 antigen-expressing human melanomas (FMX-Met, SESX) but not in low antigen-expressing tumors (LOX-L) xenografted in nude mice. The imaging index of tumor cells was positively correlated with the antigen density of the various melanoma cell lines as measured by flow cytometry. The nonspecific immunoglobulin RPC-5 of the same IgG2a subclass as 9.2.27 did not specifically localize to xenografts of melanoma. The total amount of 125I-9.2.27 accumulated in the tumor was directly correlated with tumor size. However, the specific radioactivity (cpm/g) in smaller tumors was higher than that in larger tumors. Nonspecific uptake and circulating antibody levels differed between normals and tumor-bearers. The organs of the reticuloendothelial system of normal mice accumulated more labeled antibody than did those of tumor bearers, and conversely, tumor bearers had higher levels of circulating labeled antibody in the blood than normals. The circulating labeled antibody in tumor bearers was still monomeric but had no detectable antigen-binding capacity.     

Biodistribution and radiation dose estimates for yttrium- and iodine-labeled monoclonal antibody IgG and fragments in nude mice bearing human colonic tumor xenografts

An anti-carcinoembryonic antigen murine monoclonal antibody designated NP-4, and its F(ab')2 and Fab' fragments, were coupled to the 1/1 mixture of 1-isothiocyanato-benzyl-3-methyl- and 1-methyl-3-isothiocyanato-benzyl-diethylenetriaminepentaacetic acid chelate and labeled with 111In or 88Y. Biodistribution studies in nude mice bearing a human colonic tumor xenograft were performed with these labeled conjugates, and comparisons were made to unconjugated NP-4 IgG and fragments labeled with 131I. Regardless of the labeling method, higher tumor uptake was found with the intact IgG than with the fragments, but due to faster blood clearance, tumor/blood ratios were higher for the fragments than for the IgG. Tumor uptake for the radiometal-labeled NP-4 was generally higher than the 131I-labeled NP-4. Tumor/nontumor ratios for the liver, kidney, and spleen were higher for the 111In- and 88Y-labeled NP-4 IgG than the respective radiometal-labeled fragments, but tumor/nontumor ratios for the 131I-NP-4 fragments were higher than the 131I-NP-4 IgG. Radiometal uptake in the kidney was approximately 8 and 150 times higher than the 131I-NP-4 F(ab')2 and Fab', respectively, and the clearance of radiometal activity in the kidneys was approximately 10 times slower than the radioiodine. Quantitation of 88Y or 111In activity in the femur showed 3-5%/g for the IgG and F(ab')2 and only 1-2%/g for the Fab'. The amount of radioactivity in the femur remained constant over time, and between 60 and 100% of the 88Y activity remained after flushing the core of the femur with saline, whereas 50-70% of the 111In and only 25-30% of the 131I activity remained after washing. Radiation dose estimates derived from these studies suggest that at the maximal tolerated dose 131I-NP-4 IgG would deliver 5.9 times the dose to the tumor as 90Y-labeled NP-4 IgG. 90Y-labeled fragments would not be useful due to higher doses to the kidneys than to the tumor. However, with 131I-labeled IgG and fragments there is greater flexibility to permit tumoricidal doses without excessive toxicity to the normal tissues.     

Novel synthesis and in vitro characterization of disulfide-linked ricin-monoclonal antibody conjugates devoid of galactose binding activity

The use of tumor immunotherapy using whole ricin-antibody conjugates is complicated by the nonspecific lectin activity of the ricin B-chain which leads to toxic side effects. A novel method of coupling whole intact ricin to monoclonal antibody (MoAb) is described herein, where the nonspecific binding of the ricin B-chain is blocked. The coupling was done using the bifunctional reagents S-acetylmercaptosuccinic anhydride for antibody and succinimidyl 3-(2-pyridyldithio)propionate for ricin, and this resulted in the loss of B-chain binding activity, while impairing neither the toxic potential of the A-chain nor the activity of the MoAb. The purified immunotoxins could not bind to lactose-Sepharose and were equally cytotoxic in vitro to MoAb-reactive cell lines in the presence or absence of lactose. The coupling method was suitable for six different ricin-antibody conjugates and also using ricin deglycosylated by treatment with periodate. However, the blocking of the ricin B-chain was only effective with whole IgG molecules as F(ab')2-ricin immunotoxins could, like ricin, bind to lactose-Sepharose. Ricin-antibody conjugates reduced the [3H]leucine incorporation of appropriate target cells by 50% at a concentration of 6 to 45 ng/ml, whereas nonreactive antibody immunotoxins were not toxic to the target cells at concentrations as high as 10(4) ng/ml. The specific cytotoxicity of these immunotoxins could be inhibited by the addition of unconjugated reactive MoAb; the presence of lactose or a nonreactive MoAb did not significantly affect the observed cytotoxicity. Thus, whole ricin-antibody conjugates produced in this way do not bind nonspecifically to target cells, the most important implication being that such immunotoxins should be more potent that ricin A-chain conjugates and capable of being used in vivo.     

Histochemical detection of oestrogen receptors: a progress report.

Four albumin conjugates of oestradiol. Labelled with fluorescein or peroxidase to permit visualization under light or fluorescence microscopy, were synthesized. These were used to examine the feasibility of identifying oestrogen binding in frozen section by two published histochemical techniques. In a variety of experimental tissues and human breast cancers, binding of the oestrogen conjugates was demonstrable, but it appeared nonspecific (i.e., rarely displaceable by competitor) and unrelated to oestrogen receptor (RE) status of the tissue as determined biochemically by assay with dextran-coated charcoal. Investigation of the fate of the RE through the various steps of a histochemical assay, demonstrated major losses of RE from unfixed tissue or after tissue fixation. The RE also exhibited a 10-50-fold poorer affinity for the conjugates synthesized than for oestradiol-17 beta and, at the concentrations of conjugate routinely used in histochemical assays, it seems likely that considerable nonspecific binding takes place. These factors may combine to make it (1) difficult to implement such histochemical assays and (2) unlikely that the RE is being detected.     

Limitations of radiolabeled monoclonal antibodies for localization of human neoplasms

Tumor-associated monoclonal antibodies were radiolabeled with 125I and 131I and given i.v. in pairs to 19 patients 1-26 days prior to surgical excision of primary and metastatic breast, ovarian, and gastrointestinal tumors. For individual patients each monoclonal antibody was designated as specific or nonspecific according to prior immunoperoxidase staining results on the appropriate target neoplastic tissues. Quantitation of antibody uptake was performed on resected normal and neoplastic tissues. Although good tumor:non-tumor ratios were obtained with the specific antibodies (maximal tumor:blood ratio, 35.8:1 at 12 days postadministration), the absolute amount of radiolabel detected in tumors was small (mean value of 0.015% of total injected amount per g of tumor occurring 1 day postadministration). Furthermore, both specific and nonspecific antibodies accumulated in normal lymph nodes to a significant extent (mean value of 0.0026% of total injected amount per g of tissue occurring 1 day postadministration). Knowledge of such data is essential prior to considering therapeutic uses of radiolabeled monoclonal antibodies.     

Attempts to target antitumor drugs toward opioid receptor-rich mouse tumor cells with enkephalin-ellipticinium conjugates

Human colorectal and pulmonary carcinomas have been shown to contain high levels of opioid peptides and their corresponding membrane-bound receptors. Therefore possible targeted drugs, consisting of modified enkephalins linked to cytotoxic drugs, were designed. Such conjugates were expected to be specifically internalized within opioid receptor-bearing cells. As a model to this approach, we have synthesized enkephalin-ellipticinium conjugates in which the D-Ala2-D-Leu5-enkephalin (DADLE) was coupled to the 2-nitrogen of either ellipticine or 9-hydroxyellipticine, two drugs acting through different mechanisms of cytotoxicity. These conjugates, DADLE-ellipticinium (NME) and DA-DLE-9-hydroxyellipticinium (NMHE), respectively, were previously shown to retain in vitro both opioid receptors and DNA affinities close to those of the parent compounds. In this paper, we first show that each individual moiety in the complexes remains capable of recognizing its cellular targets. Thus, pretreatment of NG108-15 cells containing delta-opioid receptors by the DADLE-ellipticinium conjugates induced a loss of opioid receptor (down-regulation), while the smaller peptide conjugates, tyrosinyl-D-alanylglycine-ellipticinium, prepared as control, do not. On the other hand, peptide-NMHE conjugates were able to induce DNA topoisomerase II-associated DNA strand breaks suggesting that they have a mode of action similar to that of their parent molecule, NMHE. We then examined whether or not these molecules could exert a specific toxicity on opioid receptor-bearing cells. However, when tested on NG108-15 tumor cells and L-fibroblasts as control, the enkephalin-ellipticinium conjugates (DADLE-NME and DADLE-NMHE) proved to be similarly more cytotoxic on both cell lines than their ellipticinium (NME and NMHE) precursors. In order to understand this apparent lack of specificity we examined the cellular accumulation and distribution of DADLE-NME by fluorescence techniques. These experiments revealed that an important intracellular overconcentration caused by a nonspecific process is probably masking the specific targeted effect of the conjugates. Hence, the project of linking DADLE to highly cytotoxic molecules which cannot cross the plasma membrane without site-directed targeting is discussed.     

Conjugates of mitomycin with anti-alpha-fetoprotein antibody: antitumor effect against alpha-fetoprotein-producing human gastric carcinoma implanted in nude mice

Conjugates of mitomycin (MMC) with an affinity-purified horse anti-human alpha-fetoprotein (AFP) antibody were prepared by direct conjugation or by indirect conjugation mediated by human serum albumin; 1a-[4-(N-succinimidoxycarbonyl)butyryl]-MMC was used in the preparation. In therapeutic experiments with athymic nude mice inoculated with the human AFP-producing gastric carcinoma OSS, the conjugates caused a greater inhibition of the tumor growth than did an unconjugated mixture of anti-AFP antibody and MMC or similar conjugates with normal horse immunoglobulin. The AFP serum level and its ratio to tumor volume were low in the group of mice treated with anti-AFP conjugate. The enhanced drug activity of the anti-AFP conjugates was not observed against human gastric carcinoma YSS (no AFP production) in nude mice; this suggests the involvement of the specific antibody-antigen interaction in the activity of the anti-AFP conjugates.     

Hydrolysis of methotrexate-immunoglobulin conjugates by liver homogenates and characterization of catabolites

Summary Methotrexate (MTX) linked to antitumor antibodies inhibits tumor growth better than free MTX, free antibody, or MTX linked to normal rabbit IgG (NRG), in spite of the less effective inhibition of the target enzyme dihydrofolate reductase (DHFR) by conjugated MTX. In addition to the demonstrated higher uptake of MTX linked to antitumor antibodies (compared with the uptake of free MTX or nonspecific IgG conjugates), a contributory factor to the superior tumor inhibitory action of MTX-IgG conjugates may be the prolonged release of active drug from the internalized conjugate. Therefore, we have investigated whether an MTX-IgG conjugate could be hydrolyzed to release free MTX or fully active MTX-containing fragments after incubation with liver homogenates and have characterized the catabolites according to the presence of free MTX and their capacity to inhibit DHFR. Catabolism was optimal at pH 4.6, activated by dithiothreitol, and inhibited by antipain and N--p-tosyl-l-lysine chloromethyl ketone, thus implicating lysosomal enzymes. Liver homogenates produced an MTX-containing, lowmolecular-weight fraction that was isolated by gel filtration. Further purification of this fraction by DEAE-cellulose chromatography gave two MTX-containing peaks, neither of which migrated as free MTX on thin-layer chromatography or inhibited DHFR more effectively than the parent conjugate. However, the presence of amino acid residues in these catabolites could contribute to their observed prolonged intracellular retention and superior antitumor action.Supported by grants [MA 9725 and MA 10008] from the Medical Research Council of Canada     

Non‐internalizing antibody–drug conjugates display potent anti‐cancer activity upon proteolytic release of monomethyl auristatin E in the subendothelial extracellular matrix

Antibody–drug conjugates (ADCs) represent a promising class of biopharmaceuticals with the potential to localize at the tumor site and improve the therapeutic index of cytotoxic drugs. While it is generally believed that ADCs need to be internalized into tumor cells in order to display optimal therapeutic activity, it has recently been shown that non‐internalizing antibodies can efficiently liberate disulfide‐linked drugs at the extracellular tumor site, leading to potent anti‐cancer activity in preclinical animal models. Here, we show that engineered variants of the F16 antibody, specific to a splice isoform of tenascin‐C, selectively localize to the subendothelial tumor extracellular matrix in three mouse models of human cancer (U87, A431, MDA‐MB‐231). A site‐specific coupling of F16 in IgG format with a monomethyl auristatin E (MMAE) derivative, featuring a valine‐citrulline dipeptide linker equipped with a self‐immolative spacer, yielded an ADC product, which cured tumor‐bearing mice at a dose of 7 mg/Kg. The observation of an efficient extracellular proteolytic cleavage of the valine‐citrulline linker was surprising, as it has generally been assumed that this peptidic structure would be selectively cleaved by cathepsin B in intracellular compartments. The products described in this article may be useful for the treatment of human malignancies, as their cognate antigen is strongly expressed in the majority of human solid tumors, lymphomas and aggressive leukemias, while being virtually undetectable in most normal adult tissues.