Biodistribution of charged 17.1A photoimmunoconjugates in a murine model of hepatic metastasis of colorectal cancer

Optimizing photodynamic therapy involves attempting to increase both the absolute tumour content of photosensitizer and the selectivity between tumour and surrounding normal tissue. One reason why photodynamic therapy has not been considered suitable for treatment of metastatic tumours in the liver, is the poor selectivity of conventional photosensitizers for tumour compared to normal liver. This report details an alternative approach to increasing this selectivity by the use of antibody-targeted photosensitizers (or photoimmunoconjugates) to target intrahepatic tumours caused by human colorectal cancer cells in the nude mouse, and explores the role of molecular charge on the tumour-targeting efficiency of macromolecules. The murine monoclonal antibody 17.1A (which recognizes an antigen expressed on HT 29 cells) was used to prepare site-specific photoimmunoconjugates with the photosensitizer chlorine6. The conjugates had either a predominant cationic or anionic charge and were injected i.v. into tumour-bearing mice. Biodistribution 3 or 24 h later was measured by extraction of tissue samples and quantitation of chlorine6 content by fluorescence spectroscopy. The photoimmunoconjugates were compared to the polylysine conjugates in an attempt to define the effect of molecular charge as well as antibody targeting. The anionic 17.1A conjugate delivered more than twice as much photosensitizer to the tumour at 3 h than other species (5 times more than the cationic 17. 1A conjugate) and had a tumour:normal liver ratio of 2.5. Tumour-to-liver ratios were greater than one for most compounds at 3 h but declined at 24 h. Tumour-to-skin ratios were high (> 38) for all conjugates but not for free chlorine6. Cationic species had a high uptake in the lungs compared to anionic species. The photoimmunoconjugates show an advantage over literature reports of other photosensitizers, which can result in tumour:normal liver ratios of less than 1.     

Pegylation of charged polymer-photosensitiser conjugates: effects on photodynamic efficacy

Conjugates between photosensitisers (PS) and charged polymeric carriers are under investigation for photodynamic therapy of cancer and may allow targeting to certain cell types or compartments in tumours. Covalent attachment of polyethylene glycol to macromolecules (pegylation) may alter their pharmacokinetics, cell type targeting, and photophysical properties. Macrophages may take up large amounts of aggregated PS, thus lessening the selectivity for cancer cells in tumours. We investigated the effect of pegylation on the uptake and phototoxicity of poly-L-lysine chlorin(e6) conjugates with either cationic or anionic charges in two cell lines, human ovarian cancer cells and mouse macrophages. The cationic conjugate after pegylation became less aggregated, consumed less oxygen and had reduced cellular uptake. However, the phototoxicity corrected for cellular uptake increased three- to five-fold. In contrast, the anionic succinylated conjugate on pegylation became more aggregated, consumed similar amounts of oxygen, and had higher cellular uptake. The anionic conjugate showed the highest relative phototoxicity towards both the cell lines (compared to the other three conjugates) and it decreased most towards the macrophages after pegylation. Pegylation reduced the amount of oxygen consumed per chlorin(e6) molecule when photosensitised cells were illuminated. These in vitro studies suggest that pegylation alters the phototoxicity of PS conjugates depending on the effect produced on the aggregation state.     

Biodistribution of charged F(ab')2 photoimmunoconjugates in a xenograft model of ovarian cancer.

The effect of charge modification of photoimmunoconjugates (PICs) on their biodistribution in a xenograft model of ovarian cancer was investigated. Chlorin(e6)c(e6) was attached site specifically to the F(ab'')2 fragment of the murine monoclonal antibody OC125, directed against human ovarian cancer cells, via poly-1-lysine linkers carrying cationic or anionic charges. Preservation of immunoreactivity was checked by enzyme-linked immunosorbent assay (ELISA). PICs were radiolabelled with 125I and compared with non-specific rabbit IgG PICs after intraperitoneal (i.p.) injection into nude mice. Samples were taken from normal organs and tumour at 3 h and 24 h. Tumour to normal 125I ratios showed that the cationic OC125F(ab'')2 PIC had the highest tumour selectivity. Ratios for c(e6) were uniformly higher than for 125I, indicating that c(e6) became separated from 125I. OC125F(ab'')2 gave highest tissue values of 125I, followed by cationic OC125F(ab'')2 PIC; other species were much lower. The amounts of c(e6) delivered per gram of tumour were much higher for cationic OC125F(ab'')2 PIC than for other species. The results indicate that cationic charge stimulates the endocytosis and lysosomal degradation of the OC125F(ab'')2-pl-c(e6) that has bound to the i.p. tumour. Positively charged PICs may have applications in the i.p. photoimmunotherapy of minimal residual ovarian cancer.     

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.     

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.     

Targeted photodynamic therapy with multiply-loaded recombinant antibody fragments

Current photodynamic therapy (PDT) of cancer is limited by inefficiencies involved in specifically targeting photosensitizers to tumors. Although antibodies are being explored as targeting vehicles, they present significant challenges, particularly in terms of pharmacokinetics and drug-coupling. We describe here a novel and effective system to covalently attach multiple photosensitizer molecules (both preclinical, pyropheophorbide-a and clinically approved, verteporfin photosensitizers) to single-chain Fvs. Further, we demonstrate that not only do the resulting photoimmunoconjugates retain photophysical functionality, they are more potent than either free photosensitizer, effectively killing tumor cells in vitro and in vivo. For example, treatment of human breast cancer xenografts with a photoimmunoconjugate comprising an anti-HER-2 scFv linked to 8-10 molecules of pyropheophorbide-a leads to significant tumor regression. These results give an insight into the important features that make scFvs good carriers for PDT drugs and provide proof of concept of our unique approach to targeted photodynamic therapy (tPDT). This promises to significantly improve on current photodynamic therapies for the treatment of cancer.     

Epidermal growth factor receptor-targeted photosensitizer selectively inhibits EGFR signaling and induces targeted phototoxicity in ovarian cancer cells

Targeted photosensitizer delivery to EGFR-expressing cells was achieved in the present study using a high purity, targeted photoimmunoconjugate (PIC). When the PDT agent, benzoporphyrin derivative monoacid ring A (BPD) was coupled to an EGFR-targeting antibody (cetuximab), we observed altered cellular localization and selective phototoxicity of EGFR-positive cells, but no phototoxicity of EGFR-negative cells. Cetuximab in the PIC formulation blocked EGF-induced activation of the EGFR and downstream signaling pathways. Our results suggest that photoimmunotargeting is a useful dual strategy for the selective destruction of cancer cells and also exerts the receptor-blocking biological function of the antibody.     

Photoimmunotherapy of human ovarian carcinoma cells ex vivo.

Photodynamic therapy is a relatively new and potentially selective experimental approach to the treatment of malignant neoplasms. Its inherent dual selectivity is reinforced by the use of photosensitizer-monoclonal antibody conjugates. The goal of this study was to evaluate the phototoxicity and selectivity of an immunoconjugate (IC) synthesized from a chlorin derivative chlorin e6-monoethylenediamine monoamide (CMA) as the photosensitizer and an anti-ovarian carcinoma monoclonal antibody OC125. Binding efficiency and specificity of the IC were determined by enzyme-linked immunosorbent assay, and specific covalent linkage of the monoclonal antibody to the photosensitizer was demonstrated by fluorescence and electrophoresis. Phototoxicity was tested against ascites or pleural fluid cells from 15 patients with ovarian and nonovarian cancers. Tumor cells from the fluid were treated with the IC at 3 microM equivalent CMA concentration and irradiated at 654 nm (lambda max CMA in IC) at 25 J/cm2 from an argon ion-pumped dye laser. Phototoxic efficacy was assayed by [3H]thymidine incorporation. Ovarian cancer cells exhibited high cytotoxicity with [3H]thymidine incorporation of 2.4 +/- 2.2%, while nonovarian cancer cells under identical conditions exhibited none to reduced cytotoxicity with [3H]thymidine incorporation of 70 +/- 54%. Using a Wilcoxon test, there was a statistically significant difference between these two groups (P less than 0.001). Dose-response curves revealed reciprocity in photosensitizer concentration and fluence. These results demonstrate that photoimmunoconjugates retain significant antigen binding specificity and affinity, are effective in the selective photochemical eradication of target cells, and merit further evaluation as photochemotherapeutic agents.     

In vivo fluorescence imaging of the transport of charged chlorin e6 conjugates in a rat orthotopic prostate tumour.

Polymeric drug conjugates are used in cancer therapy and, varying their molecular size and charge, will affect their in vivo transport and extravasation in tumours. Partitioning between tumour vasculature and tumour tissue will be of particular significance in the case of photosensitizer conjugates used in photodynamic therapy, where this partitioning can lead to different therapeutic effects. Poly-l-lysine chlorin e6conjugates (derived from polymers of average Mr 5000 and 25000) were prepared both in a cationic state and by poly-succinylation in an anionic state. A fluorescence scanning laser microscope was used to follow the pharmacokinetics of these conjugates in vivo in an orthotopic rat prostate cancer model obtained with MatLyLu cells. Fluorescence was excited with the 454-528 nm group of lines of an argon laser and a 570 nm long pass filter used to isolate the emission. Results showed that the conjugates initially bound to the walls of the vasculature, before extravasating into the tissue, and eventually increasing in fluorescence. The anionic conjugates produced tissue fluorescence faster than the cationic ones, and surprisingly, the larger Mr conjugates produced tissue fluorescence faster than the smaller ones with the same charge. These results are consistent with differences in aggregation state between conjugates.     

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.     

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.     

The development and characterisation of porphyrin isothiocyanate-monoclonal antibody conjugates for photoimmunotherapy

A promising approach to increase the specificity of photosensitizers used in photodynamic therapy has been through conjugation to monoclonal antibodies (MAb) directed against tumour-associated antigens. Many of the conjugations performed to date have relied on the activated ester method, which can lead to impure conjugate preparations and antibody crosslinking. Here, we report the development of photosensitizer-MAb conjugates utilising two porphyrin isothiocyanates. The presence of a single reactive isothiocyanate allowed facile conjugation to MAb FSP 77 and 17.1A directed against internalizing antigens, and MAb 35A7 that binds to a non-internalizing antigen. The photosensitizer-MAb conjugates substituted with 1-3 mol of photosensitizer were characterised in vitro. No appreciable loss of immunoreactivity was observed and binding specificity was comparable to that of the unconjugated MAb. Substitution with photosensitizer had a minimal effect on antibody biodistribution in vivo for the majority of the conjugates, although a decreased serum half-life was observed using a cationic photosensitizer at the higher loading ratios. Tumour-to-normal tissue ratios as high as 33.5 were observed using MAb 35A7 conjugates. The internalizing conjugate showed a higher level of phototoxicity as compared with the non-internalizing reagent, using a cell line engineered to express both target antigens. These data demonstrate the applicability of the isothiocyanate group for the development of high-quality conjugates, and the use of internalizing MAb to significantly increase the photodynamic efficiency of conjugates during photoimmunotherapy.     

Specific targeting of chlorambucil to tumors with the use of monoclonal antibodies

The concept of attaching cytotoxic drugs, such as the alkylating agent chlorambucil (CBL), to "tumor-specific" antibodies for the treatment of cancer is attractive, inasmuch as the specificity of CBL could be increased and its systemic toxicity reduced. To this end, CBL was activated by N-hydroxysuccinimide to produce an active ester derivative that was covalently coupled to monoclonal antibodies reactive with murine cell surface antigens. Up to 30 molecules of CBL were specifically bound per molecule of antibody, without impairing the alkylating activity of CBL and with minimal loss of antibody activity. The in vitro cytotoxicity of the conjugate was tested by the inhibition in [3H]thymidine incorporation into tumor cells, which demonstrated the conjugate to be specifically cytotoxic toward antibody-reactive cell lines, having more activity than the free drug. In vivo treatment of (C57BL/6 X BALB/c)F1 mice bearing a murine thymoma with CBL-antibody conjugates gave prolonged survival times and greater inhibition of growth of established tumors than was obtained with free antibody or CBL alone. The study is one of the first examples of the greater toxicity of a drug coupled to antibody, inasmuch as most drugs when coupled to antibody lose activity. CBL-monoclonal antibody conjugates may, therefore, provide a means of specifically attacking tumors, which could be therapeutically useful.     

Targeted sonodynamic therapy of cancer using a photosensitizer conjugated with antibody against carcinoembryonic antigen

The goal of this study was to develop a strategy for the selective destruction of cancer cells by ultrasonic irradiation in the presence of an antibody-conjugated photosensitizer. To this end, a photoimmunoconjugate (PIC) was prepared between ATX-70, a photosensitizer of a gallium-porphyrin analogue, and F11-39, a high affinity monoclonal antibody (MAb) against carcinoembryonic antigen (CEA), which is often overexpressed in various carcinoma cells. This conjugate, designated F39/ATX-70, retained immunoreactivity against purified CEA and CEA-expressing cells as determined by enzyme-linked immunosorbent assay, flow cytometry and immunofluorescence microscopic analysis. The cytotoxicity of F39/ATX-70 against CEA-expressing human gastric carcinoma cells in vitro was found to be greater than that of ATX-70 when applied in combination with ultrasound irradiation. When in vivo anti-tumor effects in a mouse xenograft model were assessed, intravenous administration of F39/ATX-70 followed by ultrasonic irradiation produced a marked growth inhibition of tumor compared with irradiation alone or irradiation after administration of ATX-70. These results suggest that the PIC between anti-CEA MAb and ATX-70 may have applications in sonodynamic therapy where destruction of CEA-expressing tumor is required.     

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.     

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.     

Active antiviral T-lymphocyte response can be redirected against tumor cells by antitumor antibody x MHC/viral peptide conjugates.

PURPOSE: To redirect an ongoing antiviral T-cell response against tumor cells in vivo, we evaluated conjugates consisting of antitumor antibody fragments coupled to class I MHC molecules loaded with immunodominant viral peptides. EXPERIMENTAL DESIGN: First, lymphochoriomeningitis virus (LCMV)-infected C57BL/6 mice were s.c. grafted on the right flank with carcinoembryonic antigen (CEA)-transfected MC38 colon carcinoma cells precoated with anti-CEA x H-2D(b)/GP33 LCMV peptide conjugate and on the left flank with the same cells precoated with control anti-CEA F(ab')(2) fragments. Second, influenza virus-infected mice were injected i.v., to induce lung metastases, with HER2-transfected B16F10 cells, coated with either anti-HER2 x H-2D(b)/NP366 influenza peptide conjugates, or anti-HER2 F(ab')(2) fragments alone, or intact anti-HER2 monoclonal antibody. Third, systemic injections of anti-CEA x H-2D(b) conjugates with covalently cross-linked GP33 peptides were tested for the growth inhibition of MC38-CEA(+) cells, s.c. grafted in LCMV-infected mice. RESULTS: In the LCMV-infected mice, five of the six grafts with conjugate-precoated MC38-CEA(+) cells did not develop into tumors, whereas all grafts with F(ab')(2)-precoated MC38-CEA(+) cells did so (P = 0.0022). In influenza virus-infected mice, the group injected with cells precoated with specific conjugate had seven times less lung metastases than control groups (P = 0.0022 and P = 0.013). Most importantly, systemic injection in LCMV-infected mice of anti-CEA x H-2D(b)/cross-linked GP33 conjugates completely abolished tumor growth in four of five mice, whereas the same tumor grew in all five control mice (P = 0.016). CONCLUSION: The results show that a physiologic T-cell antiviral response in immunocompetent mice can be redirected against tumor cells by the use of antitumor antibody x MHC/viral peptide conjugates.     

The specific photodynamic effects of monoclonal antibodies conjugated with hematoporphyrin derivative on gastric cancer in vitro and in vivo

Murine monoclonal antibody (MoAb) BB4.3, raised against the human gastric cancer cell line BGC823, was puriffied with Protein A-Sepharose CL-4B affinity chromatography and identified as IgG2a. It was then conjugated with a hematoporphyrin derivative (HPD) by using carbodiimide. The qualitative analysis of this conjugate showed that the amount of free HPD was negligible and there were no IgG aggregates among the conjugates. The conjugate retained both the antibody and photochemical activity of HPD. In vitro, the phototoxic effect of this HPD-BB4.3 conjugate on target cells was about 15 times higher than that of free HPD. The quality of selective photocytotoxicity was proven by the greater cytotoxicity this conjugate showed than that of corresponding normal mouse IgG (NIgG) conjugated with HPD. It showed less cytotoxicity to colon cancer cell line B-80 (negative reaction to MoAb BB4.3) than to BGC823. Moreover, its cytotoxicity to BGC823 cells could be blocked specifically by excess BB4.3 antibody, but not by another MoAb 3G9, which combines with BGC823 at different binding sites from MoAb BB4.3. Nude mice inoculated with 2 × 10² BGC823 cells were given HPD-BB4.3, HPD, HPD-NIgG, HPD plus BB4.3 and PBS, respectively then exposed to light. Four out of six animals treated with the HPD-BB4.3 conjugate remained tumor-free for a long period. Although two developed tumors, there was a significant difference between the HPD-BB4.3-treated group and all the control groups in tumor induction time, tumor growth rate, and survival time (p<0.001). The HPD-BB4.3 conjugate inhibited the growth of established tumors by more than 40% in comparison with control groups (p<0.05).     

Preparation and in vitro cytotoxicity of a methotrexate-anti-MM46 monoclonal antibody conjugate via an oligopeptide spacer

A method was developed by which conjugates of methotrexate (MTX) with antibody were prepared via an oligopeptide spacer which, after internalization of the conjugates into the target cells, would be cleaved by lysosomal enzymes to liberate MTX or its derivative(s) for entry of the drug into the cytoplasm through the lysosomal membrane. The conjugate of MTX with a monoclonal antibody (MAb) (aMM46) to mouse mammary carcinoma MM46 cells prepared by this method via Leu-Ala-Leu-Ala showed potent, antibody-directed cytotoxicity through lysosomal degradation of the conjugate, most likely at the tetrapeptide spacer. This was supported by the following observations: (a) the cytotoxicity of the aMM46 conjugate was more potent than that of the corresponding normal gamma-globulin conjugate, the antibody alone not being cytotoxic; (b) the conjugate retained its potent cytotoxicity to MM46 cells even after removal, by hydroxylamine treatment, of a less stably bound MTX derivative which might have been released extracellularly and have caused non-specific cytotoxicity by entering the cells via the membrane active transport system for MTX; (c) the cytotoxicity was not inhibited by thiamine pyrophosphate, an inhibitor of the MTX transport system; (d) the cytotoxicity was inhibited significantly with ammonium chloride, which inactivates lysosomal enzymes by raising the pH; and (e) the cleavability of the Leu-Ala-Leu-Ala spacer by the lysosomal enzymes was verified by using bio-undegradable poly(D-lysine) and biodegradable poly(L-lysine) as the lysosomotropic macromolecular carriers: unlike the poly(L-lysine) counterpart, the direct MTX-poly(D-lysine) conjugate showed very weak cytotoxicity while the tetrapeptide-mediated conjugate of MTX with poly(D-lysine) exhibited potent cytotoxicity.     

Folate receptor-targeted immunotherapy: induction of humoral and cellular immunity against hapten-decorated cancer cells

We previously exploited the frequent overexpression of folate receptors on cancer cells to decorate malignant cell surfaces selectively with folate-hapten conjugates. In antihapten-immunized hosts, this targeted localization of foreign haptens to tumor cells led to rapid accumulation of autologous antihapten IgG, which in turn yielded potent antitumor activity upon stimulation with cytokines (IL-2, IFN-alpha). In an effort to understand the effector mechanisms responsible for tumor regression, we have now investigated the involvement of both humoral and cellular immune components in the tumor destruction process. We report that the dependence of therapeutic efficacy on folate-hapten concentration is bimodal, suggesting that the conjugate must bridge between a cell surface FR and an antihapten IgG in order to mediate killing. Studies with cancer cells in vitro further demonstrate that folate-fluorescein-marked tumor cells are killed primarily by antibody-dependent cellular cytotoxicity and phagocytosis, with no contribution from complement-dependent mechanisms. Investigations of specific immune cell involvement also reveal that asialo-GM1(+)-natural killer cells, macrophages, CD4+ T cells and CD8+ T cells contribute significantly to recognition/removal of the cancer mass, and that elimination of these cell types markedly compromises the therapy. Because the initial antibody-dependent stage of tumor cell killing is shown to lead to a long-term antibody-independent cellular immunity that involves both CD4+ and CD8+ T cells, we propose that F(c) receptor-expressing immune cells not only initiate destruction of the IgG-marked tumor cells, but also participate in presentation of endogenous tumor antigens in a manner that leads to long-term cellular immunity.