Doxorubicin: monoclonal antibody conjugate for therapy of human cervical carcinoma.

Doxorubicin (Dox) was conjugated via a dextran linker to the F(ab')2 fragment of monoclonal antibody (MAb) 1H10 which recognizes an antigen expressed on the surface of human cervical carcinoma cells and tissues. Drug-antibody conjugates (1H10-Dox) with a molar ratio of Dox to MAb ranging from 40:1 to 60:1 retained antigen-binding and pharmacological activities. Anti-tumor activity of the conjugate in vitro was evaluated by measuring inhibition of [5-3H]-uridine incorporation into cellular RNA. 1H10-Dox was found to be 30 times more toxic to cervical tumor cells than a control MAb-Dox conjugate and 150 times more potent than Dox coupled to dextran. In addition, 1H10-Dox was less toxic to antigen-negative cells in vitro, suggesting that 1H10-Dox killing of cervical carcinoma cells was antibody-mediated. 125I-labeled 1H10-Dox preferentially localized in solid human cervical carcinoma xenografts in athymic mice with tumor-to-blood ratios of 1H10-Dox reaching 17.9 after 24 hr and 32.8 after 48 hr. Treatment of athymic mice bearing human cervical tumors with 1H10-Dox resulted in a dose-dependent inhibition of tumor growth. Multiple administrations of 1H10-Dox at a dose corresponding to 20 micrograms doxorubicin significantly suppressed the growth of human cervical tumors in nude mice without significant side effects (weight loss), and this suppression was antibody specific. Both i.p. and i.v. administration of 1H10-Dox were found to be equally effective. Our results suggest that 1H10-Dox may be useful for the treatment of human cervical carcinoma.     

Monovalent immunotoxin containing truncated form of Pseudomonas exotoxin as potent antitumor agent.

Recombinant truncated forms of Pseudomonas exotoxin A that lack the cell binding domain of Pseudomonas exotoxin A were coupled to an F(ab') fragment of a monoclonal antibody HB21 directed against the human transferrin receptor. One of these was NlysPE40. The other, NlysPE38QQR, has two amino groups on residues near the NH2-terminus and has no amino groups near the COOH-terminus. The proteins were linked by a stable thioether bond that connected the sulfhydryl group present in the hinge region of the antibody fragment to an amino group on the toxin. The F(ab')-PE40 immunotoxin, containing NlysPE40, exhibited potent cytotoxic activity on human carcinoma cell lines with a concentration of immunotoxin at which isotope incorporation falls by 50% when compared to nontreated cells (ID50) of 5.3 pM (0.5 ng/ml) on both the epidermoid carcinoma A431 and on the colon carcinoma Colo205. Immunotoxins made with whole antibody were considerably less active, with an ID50 of 15.9 pM (3.1 ng/ml) on these cell lines. F(ab')-PE38QQR, the immunotoxin containing NlysPE38QQR, was found to be the most active agent with an ID50 of 1.05 pM (0.1 ng/ml) on A431 cells. The greater cytotoxicity of immunotoxins containing fragmented antibody was probably due to the higher binding affinity of F(ab') conjugates in comparison to whole antibody conjugates to the transferrin receptor. The increase in cytotoxic activity of the immunotoxin made with NlysPE38QQR than that with NlysPE40 may reflect selective coupling of the toxin through NH2-terminal amino groups. The monovalent and divalent immunotoxins had dose-dependent antitumor effects on human epidermoid carcinoma xenografts in nude mice. A431 tumors completely regressed in all animals at a total dose of 105 pmol (10 micrograms) of F(ab')-PE38QQR and of 154 pmol (30 micrograms) of IgG-PE38QQR. Furthermore, the F(ab') immunotoxin was less toxic to mice than the conjugate containing IgG (840 pmol or 80 micrograms of total dose causing measurable adverse effects versus 208 pmol or 40 micrograms, respectively). Thus, a truncated Pseudomonas exotoxin A molecule coupled to the F(ab') fragment of an antibody is more active and less toxic in mice than an immunotoxin made with a whole antibody. Therefore, the therapeutic index for the monovalent immunotoxin is about four times better than that for the divalent immunotoxin.     

Antitumor effects of B3-PE and B3-LysPE40 in a nude mouse model of human breast cancer and the evaluation of B3-PE toxicity in monkeys.

B3 is a tumor-reactive monoclonal antibody (mAb) that binds to a limited number of normal tissues. Immunotoxins made with B3 coupled to either Pseudomonas exotoxin (PE) or recombinant forms of PE with a deletion of the cell-binding domain (LysPE40) have been shown to cause complete tumor regression in nude mice bearing a rapidly growing A431 (L. H. Pai et al., Proc. Natl. Acad. Sci. USA, 88: 3358-3362, 1991) human epidermoid carcinoma. In this study we show that an immunotoxin composed of mAb B3 when chemically coupled to LysPE40 (B3-LysPE40) led to complete regression of a slowly growing breast cancer, MCF-7, in nude mice when given i.v. every other day for five doses. mAb B3 coupled to native PE also produced significant regression of the MCF-7 tumor. The reactivity of mAb B3 was evaluated using an immunohistochemical method on the two responsive tumors, MCF-7 and A431, and compared with a typical human colon carcinoma specimen that has B3 antigen on its surface. The results showed that both A431 and MCF-7 xenograft tumors have similar reactivity to B3 when compared with the human colon carcinoma specimen. To evaluate the toxicity of B3-PE in primates, Cynomolgus monkeys received escalating doses of B3-PE i.v. on Days 1, 3, and 5. Based on antibody localization studies using frozen sections of normal human and monkey tissue, gastric, trachea, and bladder mucosal injury could have occurred. However, no clinical signs of injury or histological damage to these organs were seen at the doses administered. Chemical hepatitis due to PE was transient and well tolerated at doses up to 50 micrograms/kg for three doses. The lethal dose was about 100 micrograms/kg, and the cause of death was liver necrosis, as shown by necropsy. We conclude that mAb B3, when coupled to PE40 or PE, can produce strong antitumor activity in vivo. The similar level of reactivity of the B3 antibody in our tumor models with a surgical specimen of a human colon carcinoma and the toxicity study in monkeys indicate that therapeutic doses of B3-PE and B3-LysPE40 can be delivered without causing toxicity to normal organs that express B3 antigen. Although both B3-PE and B3-LysPE40 have antitumor activity in nude mice bearing a human xenograft, B3-LysPE40 is better tolerated and should be further evaluated as a therapeutic agent for cancer patients.     

Antitumor activity of SS(dsFv)PE38 and SS1(dsFv)PE38, recombinant antimesothelin immunotoxins against human gynecologic cancers grown in organotypic culture in vitro.

PURPOSE: Mesothelin, a cell surface glycoprotein overexpressed in ovarian cancer, mesotheliomas, and some squamous cell carcinomas, is an attractive candidate for targeted therapy because it is not shed in significant amounts into the bloodstream and is not present in significant amounts on normal human tissues except for mesothelial cells. The objective of this study was to determine the antitumor activity of SS1(dsFv)PE38, a recombinant antimesothelin immunotoxin, against human gynecologic tumors grown in short-term culture in vitro. EXPERIMENTAL DESIGN: Tumor cells obtained from primary cultures of five ovarian and one cervical tumor were mixed with an equal proportion of NIH-3T3 fibroblasts and plated inside collagen gels in tissue culture plates. After 4-7 days of growth, these organotypic cultures were treated with media alone, SS1(dsFv)PE38, and a control immunotoxin RFB4(dsFv)PE38, which targets the CD22 antigen not present on gynecologic tumors, every other day x 3. The organotypic culture gels were then formalin fixed, paraffin embedded, and evaluated for immunotoxin sensitivity using light microscopic examination of H&E-stained slides and also evaluated for apoptosis using the terminal deoxynucleotidyl transferase-mediated nick end labeling assay. RESULTS: Tumors expressing mesothelin showed a significant dose-dependent sensitivity to SS1(dsFv)PE38 even at concentrations as low as 1 ng/ml, whereas no antitumor activity was seen at 100 ng/ml in tumors that did not express mesothelin. This activity was specifically attributable to mesothelin targeting because RFB4 (dsFv)-PE38 had no activity against mesothelin-expressing tumors. CONCLUSIONS: These results demonstrate that ovarian and cervical tumor cells obtained from patients can be grown in short-term culture using an organotypic culture model. Our results also show low concentrations of an immunotoxin targeting mesothelin is cytotoxic to mesothelin-expressing human tumors by inducing apoptosis.     

Pharmacokinetics and antitumor activity of a bivalent disulfide-stabilized Fv immunotoxin with improved antigen binding to erbB2.

We have generated a stable bivalent Fv molecule [(dsFv)2] of the anti-erbB2 monoclonal antibody e23 in which the V(H) and V(L) domains of the Fv are linked to each other by a disulfide bond and the two Fvs are connected by a 15-amino acid linker (T. K. Bera et al., J. Mol. Biol., 281: 475-483, 1998). The e23 (dsFv)2 molecule is linked to a truncated form of Pseudomonas exotoxin (PE38) to generate a bivalent disulfide-stabilized immunotoxin e23 (dsFv)2-PE38. Compared to the monovalent immunotoxin, the (dsFv)2 immunotoxin showed greatly increased cytotoxicity to four cancer cell lines expressing low levels of erbB2 but not to four other cell lines with high erbB2 expression. e23 (dsFv)2-PE38 was administered i.v. to mice, and its half-life was determined. The t(1/2)alpha and t(1/2)beta were 20 and 325 min, respectively, whereas the corresponding values for the monovalent dsFv immunotoxin were shorter, 6 and 52 min. The antitumor activities of the monovalent and bivalent immunotoxin were compared using mice bearing A431 tumors. Despite the fact that e23 (dsFv)2-PE38 was 13-fold more active than e23 dsFv-PE38 on A431 cells in cell culture, its antitumor activity in mice was <2-fold that of the monovalent immunotoxin. These data show that a large increase in avidity does not always lead to an increase in cytotoxic activity. Furthermore, in one of the cases in which cytotoxic activity in vitro was greatly enhanced, there was only a small increase in antitumor activity.     

An immunotoxin composed of monoclonal anti-Thy 1.1 antibody and a ribosome-inactivating protein from Saponaria officinalis: potent antitumor effects in vitro and in vivo

The ribosome-inactivating protein saporin, from Saponaria officinalis, was coupled by a disulfide bond to monoclonal anti-Thy 1.1 antibody (OX7) and to its F(ab')2 fragment. The immunotoxins were at least as toxic as the plant toxin ricin to the Thy 1.1-expressing cell lines AKR-A and BW5147 in tissue culture. They reduced the rate at which the cells incorporated [3H]leucine into protein by 50% at cell concentrations of 1.5-3 X 10(-11) and 3 X 10(-12) M, respectively. The toxic effect was specific. No toxicity was seen when the immunotoxins were applied to Thy 1.2-expressing EL 4 lymphoma cells at 3 X 10(-8) M, and a control immunotoxin made from an antibody (R10) of irrelevant specificity was without effect on AKA-A cells. Further, the treatment of spleen cells from AKR mice with OX7-saporin at 10(-8) M abolished their response to the T-lymphocyte mitogen concanavalin A, without impairing their response to the B-lymphocyte mitogen lipopolysaccharide. A single iv injection of OX7-saporin into nu/nu randombred mice bearing peritoneal AKR-A lymphoma cells prolonged the survival time of the animals by an extent corresponding to that expected if 99.999% of the tumor cells had been eradicated by the immunotoxin. None of the control materials (unconjugated OX7, unconjugated saporin, OX7 plus saporin, or R10-saporin) delayed tumor growth. The OX7 F(ab')2-saporin conjugate was also highly effective as an antitumor agent, although significantly less so than the conjugate made with intact OX7. Unexpectedly, the acute toxicity of saporin to mice (median lethal dose = 6.8 mg/kg) was elevated eightfold to sixteenfold by conjugation to OX7, R10, or OX7 F(ab')2. Histologic examination of recipients of the immunotoxin revealed gross damage to hepatic parenchymal cells and to the white pulp of the spleen, neither of which was caused by unconjugated saporin. Ricin A-chain coupled to OX7 antibody was one hundredfold to one thousandfold less effective than OX7-saporin as an antitumor agent in vivo, although the two immunotoxins were equally cytotoxic to AKR-A cells in vitro.     

Genistein,a soy isoflavone,induces glutathione peroxidase in the human prostate cancer cell lines LNCaP and PC-3

Treatment of malignant brain tumors remains a clinical challenge. New treatment modalities are under investigation and among these are intratumoral infusion of immunotoxins that bind to specific cell surface molecules on the malignant cells. We have compared the efficacy of the 425.3-PE immunotoxin (which targets the epidermal growth factor [EGF] receptor) with the well-known immunotoxin Tfn-CRM107 (which targets the transferrin receptor), for the treatment of subcutaneous and intracranial human gliomas in nude animals. Bolus intratumoral administration of 1 microg Tfn-CRM107 or 425.3-PE into sc U87Mg tumors in nude mice reduced the tumor volume to 29 and 79%, respectively, of that in the control group 18 days after start of treatment. Higher doses of Tfn-CRM107 were toxic to the animals, whereas 425.3-PE was tolerated, with a dose-response relationship of up to 8 microg, a dose that reduced the tumor volume to 2% of control. In nude rats, treatment of intracerebral U87Mg tumors with Tfn-CRM107 proved ineffective and doses above 10 ng/animal were toxic to tumor-bearing rats. In contrast, intratumoral administration of 4 microg 425.3-PE increased symptom-free survival from 23 days to 40 days, with 2/9 surviving more than 90 days. We have recently shown that immunodeficient rats inoculated intracerebrally with precultured glioblastoma biopsy specimens develop highly infiltrative brain tumors. Direct interstitial infusion of immunotoxins into such tumors reduced the number of animals with detectable tumors at autopsy after 3 months, from 8/9 in the control animals to 4/6 and 2/6 in animals treated with Tfn-CRM107 and 425.3-PE, respectively. In conclusion, the anti-EGF receptor immunotoxin 425.3-PE exhibited promising efficacy, comparable to or better than that of Tfn-CRM107, an immunotoxin that in early clinical trials has been found to give responses in patients with brain tumors.     

Localization of radiolabelled F(ab')2 fragments of monoclonal antibodies in nude mice bearing intraperitoneally growing human ovarian cancer xenografts

The biodistribution and pharmacokinetics of 2 monoclonal antibodies (MAbs) specific for ovarian carcinoma, OC125 and OV-TL3, were studied in nude mice bearing intraperitoneally (i.p.) growing human ovarian carcinoma xenografts of NIH:OVCAR-3. The ovarian carcinoma xenografts grew as non-adherent cells in ascites and as solid implants in the peritoneal cavity of injected mice. The biodistribution and pharmacokinetics were determined by measurement of radioactivity in tumor masses, ascites, blood and other tissues after intravenous (i.v.) and i.p. injection of radioiodinated F(ab')2 fragments of MAbs. The specificity of the observed tumor localization was then evaluated by comparing the uptake of the anti-ovarian carcinoma antibodies OC125 and OV-TL3 with the uptake of a radioiodinated non-ovarian carcinoma-specific MAb A2C6. The results of the study indicate that uptake of the anti-ovarian carcinoma antibodies was highest in the non-adherent tumor cells in the ascites after i.p. injection. The observed uptake was 85% injected dose/g for OV-TL3 and 22% injected dose/g for OC125. This compares to the observed antibody uptake of 9% injected dose/g for OV-TL3 and less than 1% injected dose/g for OC125 in solid tumor masses after i.p. injection. After i.v. injection, uptake of OC125 and OV-TL3 was less than 3% injected dose/g, both for nonadherent tumor cells and for solid tumor masses. The data support the conclusion that OV-TL3 is superior to OC125 and that i.p. administration of radiolabelled MAb F(ab')2 fragments is superior to their i.v. administration for immunotherapy of ovarian carcinoma.     

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.     

Phase I study of monoclonal antibody-ricin A chain immunotoxin XomaZyme-791 in patients with metastatic colon cancer

Monoclonal antibody 791T/36, recognizing a Mr 72,000 antigen on the surface of colon carcinoma cells, has been used to construct an immunotoxin by conjugating to it the ribosomal inhibitor protein, ricin toxin A chain. The antibody 791T/36 has been shown to bind to membranes of freshly disaggregated tumor cells from human colon tumors, and to localize in tumors in vivo. Subacute toxicology testing in rats receiving immunotoxin i.v. showed, at highest doses, weight loss, decreased serum albumin, and hepatocyte vacuolization without elevation in liver function tests. A Phase I dose escalation study was carried out in which 17 patients with metastatic colorectal cancer were treated with doses of immunotoxin ranging from 0.02 to 0.2 mg/kg/day in 1-h i.v. infusions for a 5-day course. Side-effects included a composite of signs and symptoms thought to be generic to ricin A chain immunotoxins, including decreased serum albumin, mild fever, and flu-like symptoms, all being reversible. Two additional findings, reversible proteinuria and mental status changes, were also noted which may be characteristic of this immunotoxin. By 10-20 days after therapy, most patients developed IgM and IgG antibodies against both the ricin toxin A chain and the immunoglobulin portion of the immunotoxin, which were asymptomatic. A strong anticombining site antibody response was seen. Biological activity manifest as mixed tumor regression was seen in five patients.     

Chemoimmunotoxin therapy against a human colon tumor (HT-29) xenografted into nude mice

The efficacy of intracavitary chemoimmunotoxin therapy for cancer treatment was evaluated using the human colon carcinoma (HT-29) which had been xenografted i.p. into nude mice. Mice bearing HT-29 were treated with an immunotoxin consisting of the monoclonal antibody OVB3 coupled to Pseudomonas exotoxin (OVB3-PE), with cyclophosphamide (Cy), or with both OVB3-PE plus Cy. Mice given injections i.p. of 3 x 10(6) HT-29 ascites cells developed a localized disease that presented as both malignant ascites and solid tumor confined to the peritoneal cavity. All mice died within 30 to 40 days. Mice that received either three or six injections of OVB3-PE at a dose of 0.5 micrograms every other day beginning 3 days post-tumor inoculation exhibited significantly increased median survival times (MSTs) (P = 0.002) of 62 and 68 days, respectively, as compared to a MST of 33 days for the controls. OVB3 alone or an irrelevant monoclonal antibody conjugated to PE exhibited no antitumor activity. The therapeutic effects of the immunotoxin could be blocked by giving a large amount of unconjugated OVB3 at the same time. Treatment of mice with Cy alone at the maximal tolerated dose (250 mg/kg) on Days 10 and 17 after tumor inoculation increased the MST from 33 days to 54 days. The maximum tolerated dose could be increased to 300 mg/kg per injection if the Cy treatment was preceded by 100 mg/kg of S-2-(3-aminopropylamino)ethyl phosphorothioic acid (WR-2721), a sulfhydryl compound that selectively protects normal tissue against the toxicity of radiation and alkylating agents. Cy plus WR-2721 treatment on Days 10 and 17 increased the MST from 35 to 61 days (P = 0.002). Interestingly, groups of mice that received either two, four, or seven treatments of OVB3-PE following Cy plus WR-2721 therapy exhibited a further increase (P less than 0.002) in MSTs to 81, 87, and 96 days, respectively. Thus, the combination of cytoreductive chemotherapy with the OVB3-PE was significantly more effective for the intracavitary treatment of established HT-29 colon cancer xenografts than either chemotherapy or immunotoxin therapy alone.     

Antitumor properties of an anti-idiotypic monoclonal antibody in relation to N-glycolyl-containing gangliosides

We examined the antitumor effects of 1E10 monoclonal antibody, an anti-idiotypic IgG to an IgM monoclonal antibody, named P3, that reacts specifically with N-glycolyl-containing gangliosides and also recognizes antigens in human breast and melanoma tumors. Two murine tumor cell lines positive for the P3 antibody, F3II mammary carcinoma (BALB/c) and B16 melanoma (C57BL/6), were employed. In BALB/c mice, vaccination with several i.p. doses at 14-day intervals of 50 microgram of 1E10 coupled to keyhole limpet hemocyanin in Freund's adjuvant, significantly reduced s.c. tumor growth of F3II carcinoma cells and the number of spontaneous lung metastases. Also, the effect of 1E10 as a biological response modifier on tumor lung colonization was evaluated in C57BL/6 mice injected i.v. with B16 melanoma cells. Interestingly, i.v. administration of 10 microgram of uncoupled 1E10 antibody, 10-14 days after inoculation of B16 cells, dramatically reduced the number of experimental metastases in comparison with lungs from mice treated with an irrelevant IgG. The present data suggest that this 'non-internal image' anti-idiotypic monoclonal antibody may activate more than one mechanism of antitumor response against melanoma and mammary tumor cells.     

Human T-lymphocytes targeted against an established human ovarian carcinoma with a bispecific F(ab')2 antibody prolong host survival in a murine xenograft model

A bispecific F(ab')2 fragment with anti-CD3 and antitumor specificity was used to target activated human peripheral blood lymphocytes (PBL) against OVCAR-3 human ovarian carcinoma cells growing i.p. in athymic mice. Mice were given injections of OVCAR-3 cells on day 0 and treated with i.p. injections of activated PBL coated with the [anti-CD3 (TR66) x antitumor (MOv18)] bispecific F(ab')2 on day 4, using an approximate effector:target ratio of 1:1. Treatment was evaluated for the ability either to block tumor growth at 15 days or to prolong survival of tumor-bearing mice. After 15 days, the incidence of mice with tumor growth was 20% among those given PBL coated with bispecific F(ab')2, whereas the incidence among mice untreated or treated with PBL alone or PBL with either parental antibody ranged from 80 to 94%. The mean survival time of tumor-bearing mice treated with PBL and bispecific F(ab')2 was 104 days, which was 3.5 times that of untreated mice and twice that of mice given PBL alone or PBL with either parental antibody. These results provide support for the concept that treatment of ovarian cancer patients with targeted T-cells could prove beneficial.     

A novel CD44v6 targeting antibody fragment with improved tumor-to-blood ratio

The chimeric monoclonal antibody U36 (cMAb U36) recognizes the CD44v6 antigen. Its potential as a radioimmunotargeting agent, as well as its safety, has been shown in previous studies in head and neck cancer patients. However, intact MAbs have long circulation time in the blood and tumor targeting may also be hampered due to the slow and incomplete diffusion into solid tumors. In comparison, smaller monovalent Fab' and divalent F(ab')2 fragments are expected to exhibit shorter circulating half-lives, better tumor penetration and are thus more likely to yield better imaging results. In this study, novel F(ab')2 and Fab' fragments from cMAb U36 were radiolabeled with 125I and the characteristics of the conjugates in?vitro were examined. The biodistribution of the conjugates were then evaluated in nude mice bearing CD44v6-expressing xenograft tumors. Furthermore, the penetration depth and distribution in tumor tissue was assessed by autoradiography in selected tumor samples. The in?vitro experiments showed that the conjugates were stable and had intact affinity to CD44v6. The biodistribution study demonstrated superior tumor-to-blood ratio for the novel cMAb U36 fragment 125I-F(ab')2 compared with both the intact MAb and the monovalent fragment form. Autoradiography also revealed better tumor penetration for 125I-F(ab')2. This study demonstrates that the use of antibody fragments may improve radioimmunotargeting and possibly improve the management of head and neck malignancies.     

Radiolocalization of human pancreatic tumors in athymic mice by monoclonal antibody DU-PAN 1

Monoclonal antibodies that selectively bind to pancreatic tumors may be useful in the therapy and diagnosis of pancreatic carcinoma. In this study we have examined the tumor localization of radioiodinated DU-PAN 1, a mouse monoclonal antibody that is selective for a human pancreatic cancer-associated antigen. After radiolabeling, both DU-PAN 1 intact monoclonal antibody and F(ab')2 fragments retained immunoreactivity and showed high affinity for the pancreatic tumor cell line CA13 in vitro. Paired-label biodistribution studies in nude mice bearing CA13 s.c. xenografts were performed. Mice received both 131I-labeled DU-PAN 1 immunoglobulin G2a or F(ab')2 fragment and 125I-labeled mouse myeloma immunoglobulin G2a or F(ab')2 fragment. Tumor uptake for 5-micrograms doses of DU-PAN 1 immunoglobulin ranged from 4.8 to 11.83% injected dose/g. Tumor uptake values for mice given 5-micrograms doses of DU-PAN 1 F(ab')2 ranged from 3.9 to 6.9% injected dose/g. Tumor uptakes of the respective myeloma controls were lower in all cases when compared with the DU-PAN 1 preparations. Tumor localization indices for 5-micrograms doses of DU-PAN 1 immunoglobulin were 3.0 and 24 h and 2.9 at 48 h. For 5-micrograms doses of DU-PAN 1 F(ab')2, tumor localization indices were 29.9 at 24 h and 90.0 at 48 h. In most cases, tumor:normal tissue ratios were greater than 3 at all time points, indicative of tumor selectivity for both DU-PAN 1 preparations, but the ratios were considerably higher using the DU-PAN 1 F(ab')2. The F(ab')2 fragment thus displays better tumor localization characteristics when compared with the intact immunoglobulin. Protein doses of DU-PAN 1 F(ab')2 of between 5 and 10 micrograms gave the best localization, although protein doses of up to 100 micrograms could be administered before apparent tumor saturation was seen.     

Recombinant immunotoxins directed against the c-erb-2/HER2/neu oncogene product: in vitro cytotoxicity, pharmacokinetics, and in vivo efficacy studies in xenograft models.

TAB-250 and BACH-250 are murine and human chimeric antibodies directed at the extracellular domain of the gp185c-erb-2 (HER2/neu) growth factor receptor overexpressed in a variety of tumor types, including ovarian and breast carcinoma. The ribosome-inhibiting plant toxin gelonin (rGel) was chemically coupled to both antibodies, and the resulting immunotoxins were purified and tested in vitro against human tumor cells expressing various levels of HER-2/neu and in vivo against human tumor xenograft models. The binding of both BACH-250 and BACH-250/rGel conjugate to target cells was essentially equivalent. Against SKOV-3 cells, the IC50 of BACH-250/rGel was 97 pM (17 ng/ml), whereas BACH-250 and rGel alone showed no cytotoxic effects. There was a clear correlation between expression levels of HER-2/neu and cytoimmunotoxin. Tissue distribution studies showed that the antibody and immunotoxin both concentrate 2-10-fold higher in tumors than in normal tissues, with optimal tumor uptake occurring 48-96 h after administration. Plasma clearance curves for BACH-250 and BACH-250/rGel showed terminal-phase half-lives of 26 and 72 h, respectively. In athymic mice bearing s.c. or i.p. SKOV-3 tumors, immunotoxin treatment slowed tumor growth by 99 and 94 % at days 35 and 49 after implantation, respectively, and lengthened the median survival by 40% (from 30 to 50 days) in mice bearing lethal i.p. tumors. We conclude that clinical development of BACH-250/rGel may be warranted in patients with HER2/neu-expressing malignancies.     

Targeting solid tumors via T cell receptor complementarity-determining region 3delta in an engineered antibody

Human Vdelta2 gammadelta T lymphocytes killed multiple solid tumors, even displaying comparable therapeutic efficacy with anti-tumor chemical-cis-platinum in an adoptive experiment in both nude and SCID murine model shown in present study. We previously found that T cell receptor (TCR) gammadelta recognize tumors via complementarity-determining region 3 (CDR3), briefly named as CDR3delta. Based on characteristics of specific binding of CDR3delta to tumor targets, we developed a novel tumor-targeting antibody, whose CDR3 in heavy chain is replaced by CDR3delta sequence derived from human ovarian carcinoma (OEC) infiltrating gammadelta T cells (gammadeltaTILs). This CDR3delta-grafted antibody OT3 exhibited specific binding activities to OEC line SKOV3 both in vitro and in vivo, which included specific binding to several tumor cell lines, interacting with heat shock protein (HSP) 60 and triggering ADCC against tumors in vitro, as well as displaying tumor imaging by radioisotope 99mTc-labeled antibody OT3 in vivo. Moreover, immunotoxin OT3-DT, CDR3delta-grafted antibody OT3 chemically conjugated with diphtheria toxin (DT) showed the anti-tumor effect on the growth of several solid tumors including OEC, cervix adenocarcinoma, hepatocellular carcinoma, and rectum adenocarcinoma to various extents in nude mice. Therefore, we have found and confirmed a novel therapeutic strategy for targeting solid tumors, making use of immune recognition characteristics of gammadelta T cells.     

Site-specific expression of transferrin receptor by human colon cancer cells directly correlates with eradication by antitransferrin recombinant immunotoxin

We determined the efficacy of HB21(Fv)PE40, a single-chain immunotoxin made by fusing the variable regions of a monoclonal antibody directed at the human transferrin receptor (TfR) with a truncated mutant of Pseudomonas exotoxin (PE), against metastatic human colon carcinoma KM12L4 cells growing in the liver or subcutis of nude mice. Organ-specific modulation of TfR expression was examined by immunohistochemistry and flow cytometry using anti-human CD71 antibody. KM12L4 cells expressed human TfR and were lysed in vitro by HB21(Fv)PE40 but not LMB-7 (a control immunotoxin specific for a Lewis Y-related carbohydrate antigen). KM12L4 cells growing in the liver expressed higher levels of TfR than cells growing s.c. Systemic administration of HB21(Fv)PE40 eliminated KM12L4 liver metastasis, whereas administration of LMB-7 did not. Treatment of mice with HB21(Fv)PE40 only delayed the growth of s.c. tumors. KM12L4 cells recovered from liver metastases, expressed higher levels of TfR, and were more sensitive to lysis by HB21(Fv)PE40 than KM12L4 cells recovered from s.c. tumors. Indeed, collectively, the data show that the expression level of the TfR by human colon cancer cells is modulated by the organ microenvironment which can be advantageous for the use of therapeutic immunotoxins.     

Recombinant human tumor necrosis factor-alpha: evidence of an indirect mode of antitumor activity

The antitumor activity of recombinant human tumor necrosis factor (rTNF-alpha) was examined on murine tumors in mice and in cultured cells in vitro. Mice were implanted intradermally with Meth A fibrosarcoma (Meth A) on day 0. rTNF-alpha caused tumor necrosis and inhibited the tumor growth when given i.v. on day 7 or 10, but not when given on day 3. When rTNF-alpha was given i.v. in doses of 0.1-3.2 micrograms/mouse twice a week for 3 weeks beginning on day 7 or 11, the growth of solid Meth A, Colon 26 adenocarcinoma, Colon 38 carcinoma, Sarcoma-180, and M5076 reticulum cell sarcoma tumors implanted s.c. or intradermally was markedly inhibited, and the life of the mice bearing these tumors, except M5076 reticulum cell sarcoma, was prolonged. The growth of Meth A implanted i.m. was also markedly inhibited by rTNF-alpha given i.v. However, the life of mice bearing i.p. Colon 26 adenocarcinoma, MH134 hepatoma, Sarcoma-180, and Ehrlich carcinoma was not prolonged by rTNF-alpha given i.p. nine times (days 1-9) in doses up to 1.0 or 3.2 micrograms/mouse. Only in the case of mice bearing i.p. Meth A, the life was slightly prolonged by i.p. treatment with rTNF-alpha but not by i.v. treatment. In experiments against in vitro cultured cells, rTNF-alpha did not show any direct cytotoxicity against mouse tumor cells: Meth A, Colon 26 adenocarcinoma, Colon 38 carcinoma, and Sarcoma-180, but had a cytotoxic effect against L929 mouse fibroblast. The results suggest that rTNF-alpha is a unique antitumor drug with potent necrotizing activity against solid tumors in mice, and that this activity may derive from indirect mechanisms related to the growth of tumors and not to the direct cytotoxicity of the drug.     

Enhanced therapeutic efficacy of an immunotoxin in combination with chemotherapy against an intraperitoneal human tumor xenograft in athymic mice

A mouse IgG2b anti-pan carcinoma monoclonal antibody, NR-LU-10, was shown to bind homogeneously to ascites xenografts of both ovarian and colon carcinoma. Following linkage to a highly potent holotoxin, Pseudomonas exotoxin A (PE), NR-LU-10 demonstrated high potency and selectivity in vitro (ID50 = 100 pg/ml; elimination of greater than or equal to 4.5 logs of cells). The conjugate was evaluated for therapeutic efficacy against a human colon tumor (HT-29) transplantable in the peritoneal cavity of nude mice. Beginning 3 days after HT-29 injection, mice received either three or six i.p. injections of 0.5 micrograms of unconjugated NR-LU-10 or immunotoxin conjugate (NR-LU-10/PE) every other day. Mice that received three or six treatments of NR-LU-10 alone had median survival times (MSTs) of 39 and 40 days, respectively, which did not differ significantly from the MST observed for the untreated control groups (MST = 35 days). In contrast, treatment with three or six injections of 0.5 micrograms NR-LU-10/PE exhibited significantly increased MSTs (P = 0.002) of 50 and 60 days, respectively. Coinjection of unconjugated NR-LU-10 (20 micrograms) and 0.5 micrograms of NR-LU-10/PE blocked the therapeutic effect of the immunotoxin (MST = 33 days). The therapeutic efficacy of NR-LU-10/PE was further enhanced against HT-29 when administered i.p. during and after cytoreductive chemotherapy. The i.p. administration of 300 mg/lg of cyclophosphamide plus 100 mg/kg of the chemoprotective drug, WR-2721, 10 and 17 days posttumor cell inoculation induced a significant increase in MST from 36 days to 59 days (P = 0.002). Interestingly, groups of mice that received either two, four, or seven treatments of NR-LU-10/PE following cytoreductive therapy exhibited a further significant increase (P = 0.001) in MSTs of 89, 97, and 105 days, respectively. Therefore, the use of immunotoxin therapy following cytoreductive chemotherapy significantly prolonged survival time of mice bearing the HT-29 colon tumor over that observed with chemotherapy or NR-LU-10/PE alone.