Targeting HER2

The potential of the HER2-targeting antibody trastuzumab as a radioimmunoconjugate useful for both imaging and therapy was investigated. Conjugation of trastuzumab with the acyclic bifunctional chelator CHX-A”-DTPA yielded a chelate:protein ratio of 3.4±0.3; the immunoreactivity of the antibody unaffected. Radiolabeling was efficient, routinely yielding a product with high specific activity. Tumor targeting was evaluated in mice bearing subcutaneous (s.c.) xenografts of colorectal, pancreatic, ovarian, and prostate carcinomas. High uptake of the radioimmunoconjugate, injected intravenously (i.v.), was observed in each of the models, and the highest tumor %ID/g (51.18±13.58) was obtained with the ovarian (SKOV-3) tumor xenograft. Specificity was demonstrated by the absence of uptake of ¹¹¹In-trastuzumab by melanoma (A375) s.c. xenografts and ¹¹¹In-HuIgG by s.c. LS-174T xenografts. Minimal uptake of i.v. injected ¹¹¹In-trastuzumab in normal organs was confirmed in non-tumor-bearing mice. The in vivo behavior of ¹¹¹In-trastuzumab in mice bearing intraperitoneal (i.p.) LS-174T tumors resulted in a tumor %ID/g of 130.85±273.34 at 24 h. Visualization of tumor, s.c. and i.p. xenografts, was achieved by γ-scintigraphy and PET imaging. Blood pool was evident as expected, but cleared over time. The blood pharmacokinetics of i.v. and i.p. injected ¹¹¹In-trastuzumab was determined in mice with and without tumors. The data from these in vitro and in vivo studies supported advancement of radiolabeled trastuzumab into two clinical studies, a Phase 0 imaging study in the Molecular Imaging Program of the National Cancer Institute and a Phase 1 radioimmunotherapy study at the University of Alabama.     

Targeting HER2: A report on the in vitro and in vivo pre-clinical data supporting trastuzumab as a radioimmunoconjugate for clinical trials

The potential of the HER2-targeting antibody trastuzumab as a radioimmunoconjugate useful for both imaging and therapy was investigated. Conjugation of trastuzumab with the acyclic bifunctional chelator CHX-A″-DTPA yielded a chelate:protein ratio of 3.4 ± 0.3; the immunoreactivity of the antibody unaffected. Radiolabeling was efficient, routinely yielding a product with high specific activity. Tumor targeting was evaluated in mice bearing subcutaneous (s.c.) xenografts of colorectal, pancreatic, ovarian and prostate carcinomas. High uptake of the radioimmunoconjugate, injected intravenously (i.v.), was observed in each of the models and the highest tumor %ID/g (51.18 ± 13.58) was obtained with the ovarian (SKOV-3) tumor xenograft. Specificity was demonstrated by the absence of uptake of ¹¹¹In-trastuzumab by melanoma (A375) s.c. xenografts and ¹¹¹In-HuIgG by s.c. LS-174T xenografts. Minimal uptake of i.v. injected ¹¹¹In-trastuzumab in normal organs was confirmed in non-tumor-bearing mice. The in vivo behavior of ¹¹¹In-trastuzumab in mice bearing intraperitoneal (i.p.) LS-174T tumors resulted in a tumor %ID/g of 130.85 ± 273.34 at 24 h. Visualization of tumor, s.c. and i.p. xenografts was achieved by γ-scintigraphy and PET imaging. Blood pool was evident as expected but cleared over time. The blood pharmacokinetics of i.v. and i.p. injected ¹¹¹In-trastuzumab was determined in mice with and without tumors. The data from these in vitro and in vivo studies supported advancement of radiolabeled trastuzumab into two clinical studies, a Phase 0 imaging study in the Molecular Imaging Program of the National Cancer Institute and a Phase 1 radioimmunotherapy study at the University of Alabama.Key words: monoclonal antibody, HER2, trastuzumab, radioimmunodiagnosis, radioimmunotherapy     

Intratumoral low-dose interleukin-2 induces rejection of distant solid tumour

Summary This study shows that local tumour treatment with low-dose recombinant interleukin-2 (IL-2) can mediate rejection of a large distant solid tumour. When SL2 lymphoma cells were injected intraperitoneally (i.p.) in syngeneic DBA/2 mice on day 0, 70% of these mice were cured by daily i. p. injections with 20 000 units IL-2 on days 10–14. After injecting mice with SL2 both i.p. and subcutaneously (s. c.) on the flank, 50% of the mice treated i.p. with low-dose IL-2 rejected both the i.p. tumour and the large distant s.c. tumour. In contrast, i.p. IL-2 treatment on days 10–14 cured fewer than 10% of the mice bearing only a s. c. SL2 tumour. The described IL-2 immunotherapy also caused systemic tumour rejection in mice bearing both ascitic and solid P815 mastocytoma. Thus it was shown that low-dose IL-2 can induce systemic tumour rejection, when injected at a site of tumour growth. Interleukin-2-induced rejection of s. c. SL2 tumour was highly specific, as mice that were rejecting i.p. and solid s. c. SL2 lymphoma did not reject solid P815 mastocytoma, which was injected s.c. simultaneously on the other flank. Furthermore, solid s.c. tumours consisting of mixtures of SL2 and P815 were not rejected in mice that rejected i.p. SL2 or P815. We conclude that intratumoral injections of low-dose IL-2 can enhance an ongoing weak immune reaction against the tumour resulting in systemic tumour rejection.     

Characterization of binding of four monoclonal antibodies to the human ovarian adenocarcinoma cell line HEY

Four mouse monoclonal antibodies (mAb) (10B, IgG1; 8C, IgG2a; M2A, IgG2a; M2D, IgG2b) were characterized with respect to their binding to the ovarian adenocarcinoma cell line HEY, using displacement assays and Scatchard plot analyses. The four mAb reacted with different antigens on the surface of HEY cells, with affinity constants ranging from 1 X 10(9) to 3 X 10(9) M-1. The number of binding sites per cell for each antibody was approximately 2 X 10(4). mAb 8C and M2D remained associated with the cell surface following binding to their respective antigens, while mAb 10B was rapidly internalized, with 50% of the bound mAb being lost from the cell surface during 4 h of incubation at 37 degrees C. These different binding characteristics of the mAb may influence their ability to target radioactivity and cytotoxic drugs to HEY cells.     

Pharmacokinetics and biodistribution of a new anti-episialin monoclonal antibody 139H2 in ovarian-cancer-bearing nude mice

Summary The new murine anti-episialin monoclonal antibody (mAb) 139H2 has been selected for its strong reactivity with a series of human ovarian cancer xenografts. In the present report we describe the characteristics of mAb 139H2 investigated in vitro as well as in vivo. Scatchard plot analysis using the human ovarian cancer cell line NIH:OVCAR-3 showed an affinity constant of 1 × 10⁸ M^–1 and the expression of 7 × 10⁶ antigenic sites/cell. Reactivity with OVCAR-3 xenograft tissue was intense, localized at the cell membrane, heterogeneously distributed, and mainly detectable at the apical site of the cell. Administration of radiolabelled mAb 139H2 to nude mice bearing s.c. OVCAR-3 xenografts showed specific uptake in the tumour up to 9% of the injected dose/g. The maximum uptake in the tumour was retained for 3.5 days and mAb 139H2 cleared from the tumour with a half-life of 5.5 days. The half-life in blood was 50 h and no antibody-antigen complex formation could be detected. Poor uptake and no retention in episialin-negative WiDr colon cancer xenografts demonstrated specificity. Administration of an excess of an unlabelled irrelevant mAb did not influence the uptake in the OVCAR-3 xenografts or in other tissues. In contrast, tumour uptake decreased after addition of 300 µg or more unlabelled mAb 139H2 to a tracer dose of radiolabelled mAb 139H2. The uptake of mAb 139H2 in OVCAR-3 xenografts appeared inversely related to the tumour size.Supported by the Dutch Cancer Society     

Risk Assessment on the Carcinogenic Potential of Hybridoma Cell DNA: Implications for Residual Contaminating Cellular DNA in Biological Products

The aim of this study was to evaluate the possible carcinogenic potential of residual DNA derived from immortalized and possibly tumorigenic cell lines due to activated oncogenic sequences (oncogenes). These cell lines have been used for the production of biologicals, i.e. monoclonal antibodies, lymphokines and vaccines. The authors used hybridoma DNA as a first model. For this reason experiments in two species were performed, namely in 3–4 week-old female Balb/c mice and newborn Riv:TOX rats. Doses of 250 μg DNA, derived from Balb/c hybridoma cells, were injected subcutaneously (s.c.) in 200 mice. These mice also received a s.c. injection of the solvent only (TE buffer) at another site of the back skin (negative control for local tumour development). An additional group of 50 mice was treated intraperitoneally (i.p.) with the solvent only to serve as a negative control group for possible systemic tumorigenic effects. Doses of 5 μg plasmid pPy1 DNA, containing the entire Polyoma virus genome, served as positive control and were injected s.c. and i.p. in 20 and 50 mice, respectively. Doses of 50 μg hybridoma DNA of 5 μg pPy1 DNA were injected s.c. in rats too, using nine animals per group. During the experiment, animals were observed weekly, especially for the occurrence of subcutaneous tumours at the injection sites. The mouse study was terminated after more than 2 years, the rat study after 1 year. Gross necropsy was performed on all animals and histopathological examination of grossly suspected neoplastic lesions was performed.In the mouse experiment, tumour development at the s.c. injection site of the DNA was observed in one out of 20 animals in the pPy1-treated positive control group (neurofibrosarcoma) and one out of 200 animals in the hybridoma DNA-treated group (haemangioma-like lesion). Tumour development at or near the s.c. injection site of the solvent only was observed in two out of 200 animals.In the rat study none out of nine hybridoma DNA-treated rats developed tumours at the injection site, while three out of nine rats of the positive control group, injected with the pPy1 DNA, showed local tumour development (benign and malignant soft tissue tumours).It is considered that, at the high dose and numbers of animals tested, parenteral administration of hybridoma DNA does not induce local tumour development. Furthermore, no indications were found for systemic carcinogenic potential of the hybridoma DNA used.Based on a worst case approach of our data, the oncogenic risk of 100 pg residual DNA was estimated to be 2×10⁻⁹, a value intermediate of the estimations of the WHO (1987) and the Dutch Health Council (1988) 5×10⁻¹¹and 2×10⁻⁷, respectively. Therefore, it is unlikely that the risk of 100 pg of DNA derived from other immortalized cell lines will exceed the level of generally accepted cancer risk of 10⁻⁶.     

Histological analysis of IL-2 induced regression of murine solid SL2-tumors

When DBA/2 mice are inoculated both intraperitoneally (i.p.) and subcutaneously (s.c.) with syngeneic SL2 lymphoma cells and treated i.p. on day 10–14 with 20,000 units IL-2/day, about 50% of the mice reject both the ascitic tumour and the s.c. tumour. During IL-2 therapy large areas of necrosis appear in the solid SL2 tumours between day 12 and 15. Immunohistochemical studies show that only a small number of infiltrating cells is present in the tumours. The percentage of macrophages (MHC-II⁺)in the tumours is about 1 and the percentage of T-lymphocytes (-TCR⁺) about 0.5. No differences in the numbers of infiltrating cells are seen in untreated and IL-2 treated tumour bearing mice. The tumoursurrounding infiltrate consists mainly of mononuclear cells: about 50% macrophages, 20% CD8⁺ cells, and 15% CD4⁺ cells. No tumour-infiltrating cells were found that express the IL-2 receptor.We conclude that direct cytotoxic activity of tumour infiltrating cells cannot account for the rapid occurrence of necrosis.When L3T4⁺ cells were eliminated by treating the mice with-L3T4 monoclonal antibodies before tumor inoculation and treatment with rIL-2, tumor eradication did not occur. So, L3T4⁺ helper T-cells are essential for IL-2-mediated tumour regression. Exogenous rIL-2 is not directly responsible for the induced tumour regression. A significant stagnation of intratumoural bloodflow is observed after histological analysis; yet it still needs to be determined whether this is the primary cause or consequence of the observed necrosis.Abbreviations BSA bovine serum albumin - CTL cytotoxic T-lymphocyte - FACS fluorescence activated cell sorter - HE haematoxylin and eosin - IFN interferon - IL-2 interleukin-2 - IL-2R interleukin-2 receptor - i.p. intraperitoneal(ly) - i.V. intravenous(ly) - LAK lymphokine-activated killer - MHC major histocompatibility complex - PBS phosphate buffered saline - s.c. subcutaneous(ly) - TCR T-cell receptor - TNF tumour necrosis factor     

Inhibition of rat yolk sac tumour growth in vivo by a monoclonal antibody to the retroviral molecule P15E

Summary A monoclonal mouse IgG2b antibody 19F8, directed towards a determinant on the retroviral transmembranous molecule p15E, binds selectively to certain rat tumours, including all tested yolk sac tumours, one rat colon carcinoma, one spontaneous kidney carcinoma and an adenovirus-type-9-induced rat breast tumour, as tested by antibody-dependent cellular cytotoxicity (ADCC) and immunohistochemistry. Groups of rats receiving yolk sac tumour F56 isografts intraperitoneally (i.p.) or subperitoneally (s.p.) were treated with this monoclonal antibody (mAb), 19F8, inoculated twice a week in doses of 100 µg. Parallel control groups received analogous inoculations of an isotype-matched monoclonal antibody. A significant growth inhibitory effect was observed with 19F8. In 5/10 rats isografted i.p., tumour outgrowth was completely inhibited and in the other 5 rats the outgrowth was delayed compared to the 10 rats in the control group, which all developed tumours. All rats of the control group developed large volumes of ascites, whereas the 5 rats in the therapy group with eventual tumour outgrowth had little or no ascites. In two experiments with rats carrying subperitoneal isografts and treated with the 19F8 mAb, tumour grew out in 4/5 and 5/10 rats, though growth was delayed compared to the control groups, in which 5/5 and 9/9 rats developed tumours. The tumours grew significantly more slowly in the therapy groups compared to the controls. All rats that developed tumours in the therapy groups showed an anti-idiotypic response against mAb 19F8. The single tumour-free rat in the first experiment and 1/5 tumour-free rats in the other showed no such response. The draining lymph node cells from the tumour-free animals showed a specific proliferative response to yolk sac tumour F56 cells in a mixed lymphocyte tumour cell culture (MLTC), and the MLTC-induced cells were cytotoxic to F56 but not to the natural-killer-sensitive rat T cell lymphoma G1—Tc1. The cytotoxic cell population was more than 90% CD4⁺. It is concluded that the two test systems for identification of the epitope of p15E detected by mAb 19F8 correlated well in detection of the epitope in the cells (immunohistochemistry) and at the cell surface (ADCC). It is also concluded that mAb 19F8 has a growth-inhibitory effect on yolk sac tumour F56 and that, as a result of the treatment, T cells with specificity for F56 are appearing in draining lymph nodes of tumour-free animals.     

Interleukin 21 therapy increases the density of tumor infiltrating CD8<Superscript>+ </Superscript>T cells and inhibits the growth of syngeneic tumors

Interleukin (IL)-21 is a recently discovered cytokine in early clinical development, which has shown anti-tumor activity in various animal models. In the present study, we examine the anti-tumor activity of IL-21 protein therapy in two syngeneic tumor models and its effect on the density of tumor infiltrating T cells. We treated mice bearing established subcutaneous B16 melanomas or RenCa renal cell carcinomas with intraperitoneal (i.p.) or subcutaneous (s.c.) IL-21 protein therapy and subsequently scored the densities of tumor infiltrating CD4⁺ and CD8⁺ T cells by immunohistochemistry. Whereas both routes of IL-21 administration significantly inhibited growth of small, established RenCa and B16 tumors, only s.c. therapy significantly inhibited the growth of large, established tumors. We found a greater bioavailability and significant drainage of IL-21 to regional lymph nodes following s.c. administration, which could account for the apparent increase in anti-tumor activity. Specific depletion of CD8⁺ T cells with monoclonal antibodies completely abrogated the anti-tumor activity, whereas NK1.1⁺ cell depletion did not affect tumor growth. In accordance, both routes of IL-21 administration significantly increased the density of tumor infiltrating CD8⁺ T cells in both B16 and RenCa tumors; and in the RenCa model s.c. administration of IL-21 led to a significantly higher density of tumor infiltrating CD8⁺ T cells compared to i.p. administration. The densities of CD4⁺ T cells were unchanged following IL-21 treatments. Taken together, these data demonstrate that IL-21 protein has anti-tumor activity in established syngeneic tumors, and we show that IL-21 therapy markedly increases the density of tumor infiltrating CD8⁺ T cells.     

Signaling through CD40 enhances cytotoxic T lymphocyte generation by CD8+ T cells from mice bearing large tumors

Recent studies have demonstrated the importance of CD40/CD154 (CD40L) interactions for the generation of cell-mediated antitumor immune responses. Here we show that signaling via CD40 (through the use of the activating anti-CD40 mAb, 1C10) can actually promote the in vitro generation of CTL activity by CD8⁺ splenic T cells from mice bearing a large MOPC-315 tumor. Anti-CD40 mAb had to be added at the initiation of the stimulation cultures of tumor-bearing splenic cells in order to realize fully its potentiating activity for cytotoxic T lymphocyte (CTL) generation, suggesting that signaling through CD40 is important at the inductive stage of antitumor cytotoxicity. Moreover, anti-CD40 mAb was found to enhance the expression of the B7-2 (CD86) and, to a lesser extent, the B7-1 (CD80) costimulatory molecules on B220⁺ cells (i.e., B cells), and B7-2 and, to a lesser extent, B7-1 molecules played an important role in the potentiating effect of anti-CD40 mAb for CTL generation by tumor-bearer splenic cells. Furthermore, B220⁺ cells were found to be essential for the potentiating effect of anti-CD40 mAb, as depletion of B220⁺ cells at the inductive stage completely abrogated the ability of anti-CD40 mAb to enhance CTL generation. Thus, signaling through CD40 enhances CTL generation by CD8⁺ T cells from tumor-bearing mice by a mechanism that involves the up-regulation of B7-2 and, to a lesser extent, B7-1 expression on B220⁺ cells. Received: 23 December 1998 / Accepted: 22 February 1999     

Pharmacokinetics and in vitro and in vivo anti-tumor response of an interleukin-2-human serum albumin fusion protein in mice

Purpose: Albuleukin fusion protein is a recombinant human interleukin-2 (rIL-2) genetically fused to recombinant human serum albumin (rHSA). The pharmacokinetics and pharmacologic activity of Albuleukin were examined in mice to determine whether the fusion protein had the immunomodulatory and anti-tumor properties of rIL-2 as well as a prolonged serum half-life due to the rHSA. Methods: The effect of Albuleukin on lymphocyte proliferation, IL-2 receptor binding, and release of IFN- from human NK cells were examined in vitro. For the pharmacokinetic analysis, Albuleukin and rIL-2 were administered intravenously (i.v.) and subcutaneously (s.c.) to BALB/c mice, both at a single dose of 500 g/kg. The anti-tumor properties of Albuleukin were evaluated in a Renca tumor model in BALB/c mice and in a metastatic liver model of B16F10 melanoma in C57B1/6 mice. In the Renca tumor model, BALB/c mice were dosed intraperitoneally (i.p.) and s.c. with Albuleukin on days 12, 14, 16, 19, 21, and 23 and i.p. with rIL-2 daily for two periods of 5 days (days 10–14 and 17–21). In the B16 melanoma model, C57B1/6 mice were dosed s.c. with rIL-2 twice daily or Albuleukin every 48 h for 14 days. Results: In vitro, Albuleukin induced the proliferation of primary human and mouse T cells and B cells and primary human NK cells, competed with rIL-2 for binding to the IL-2 receptors, and induced the production of IFN- from primary human NK cells. The s.c. bioavailability of Albuleukin was about 45% relative to the i.v. dose. Plasma half-life was prolonged and ranged from 6 to 8 h with Albuleukin, compared to 19–57 min with rIL-2. Total clearance of Albuleukin was about 50-fold slower than that of rIL-2 after i.v. dosing. In vivo, Albuleukin suppressed the growth of Renca tumors and induced a dense infiltration of CD4⁺ and CD8⁺ T cells. Both Albuleukin and rIL-2 significantly reduced the tumor burden in mice with hepatic B16F10 metastases. Albuleukin significantly reduced the incidence of residual macroscopic hepatic tumors, resulting in improved survival relative to controls and rIL-2. Conclusion: Results from these studies suggest that the therapeutic efficacy of rIL-2 is improved in mice by prolonging its in vivo half-life through genetic fusion to albumin. Albuleukin, the fusion protein, had pronounced anti-tumor effects in Renca and hepatic melanoma tumor models without an increase in mortality. On the basis of its preclinical effects, Albuleukin was brought to the clinic to assess its therapeutic benefit in a variety of cancers.     

Feasibility of UV-inactivated vaccinia virus in the modification of tumor cells for augmentation of their immunogenicity

Summary We compared the immunity induced by tumor cells modified with UV-inactivated purified vaccinia virus (UV-VV) and with live purified vaccinia virus (L-VV). C3H/HeN mice were inoculated i.p. with UV-VV or L-VV after whole-body irradiation with 150 rads of X-rays (priming). After 3 weeks the mice were immunized i.p. 3 times at weekly intervals with syngeneic X5563 or MH134 cells that had been adsorbed in vitro with UV-VV or infected with L-VV and subsequently irradiated with 10⁴ rads of X-rays. Then 1 week after the last immunization, the mice were challenged s.c. with X5563 viable tumor cells or challenged i.p. with MH134 viable tumor cells. The 50% lethal dose (TLD₅₀) of X5563 in mice primed and immunized with UV-VV (UV-VV group) on s.c. challenge (10^6.06) was the same as for mice treated with L-VV (L-VV group), whereas the TLD₅₀ of unprimed or nonimmunized mice (control group) was 10^2.61. The TLD₅₀ of MH134 in the UV-VV treated group on i.p. challenge (10^6.48) was similar to that of the L-VV treated group (10^6.54), while the TLD₅₀ of the control group was 10^1.00. The difference between the TLD₅₀ values of X5563 on s.c. challenge of mice primed and immunized with UV-VV or L-VV and control mice was 10^3.4. The difference between the TLD₅₀ values of MH134 on i.p. challenge of primed and immunized mice and control mice was 10^5.5. These results indicate that the in vivo helper function of UV-VV is similar to that of L-VV and that the augmenting effect of this protocol depends on the kind of tumor.     

Influence of the donors' clinical status on in vitro and in vivo tumor-cytotoxic activation of interleukin-2-exposed lymphocytes and their circulation in different organs

Summary In-vitro-generated lymphokine-activated killer (LAK) cells of BALB/c mice, bearing the syngeneic colon carcinoma C-26 for 7 days, were as efficient as those from normal mice in lysing C-26 cells whereas LAK cells from 14-day tumor-bearing and 5- and 14-day tumor-resected animals had a lower C-26 cytotoxicity. The level of C-26 lysis returned to normal values 30 days after surgery. To identify the best source of LAK cells in vivo, groups of normal mice were treated with 10⁴, 3×10⁴ or 10⁵ U/day of interleukin 2 (IL-2) for 7 days intraperitoneally (i. p.) or intravenously (i. v.) (3×10⁴ dose only). The highest lysis on C-26 was obtained from peritoneal exudate cells of mice given 3×10⁴ and 10⁵ U whereas spleen cells were lytic only when taken from mice treated with 10⁵ U IL-2. Peripheral blood lymphocytes lacked any cytotoxicity except for the group of mice which received IL-2 i. v. The kinetics of in vivo LAK activation in different organs showed a peak of anti-(C-26) lytic activity at day 5 in peritoneal exudate cells and spleen cells of mice given IL-2 for 5 days whereas administration of LAK cells alone had no effect; IL-2 plus LAK cells gave a lower peak of LAK activity as compared with IL-2 alone. A lower level of in vivo LAK activation was found in mice whose tumor was resected 5 days before; such impairment was evident even 14 days after surgery. Homing experiments were carried out with i. v. injected ⁵¹Cr-labelled LAK cells in normal or tumor-resected mice. In normal mice the highest radioactivity at 30 min was found in the lungs; liver and spleen also showed high radioactivity whereas blood had a negligible amount of radioactivity. Radioactivity declined rapidly in lungs (less than 10% after 24 h) while remaining at appreciable levels in the liver after 24 h and 48 h; spleen showed constant levels of 12%–15%. Homing of LAK cells was altered in mice receiving IL-2 i. p. for 5 days with slower and lower radioactivity peaks in the lung and higher levels in liver. In tumor-excised mice lower levels of radioactivity were found in lungs. These results show that: (a) alterations in LAK activity occur in early-tumor-resected and large-tumor-bearing animals; (b) the route of IL-2 administration is critical in LAK activation in vivo; (c) treatment with IL-2 modifies LAK homing.This study was in part supported by grant no. 87.01565.44 of the Finalized Project Oncology of CNR (Rome, Italy)     

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

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

Biodistribution of 111In-labelled engineered human antibody CTM01 (hCTM01) in ovarian cancer patients: influence of prior administration of unlabelled hCTM01

mAb hCTM01 binds a carcinoma-associated antigen, the MUC1 gene product. The antigen is also present in the circulation, and administration of ¹¹¹In-labelled hCTM01 results in the formation of immune complexes with enhanced accumulation in the liver. To avoid the unwanted effect of circulating radioactive immune complexes, a strategy to remove the circulating antigen was investigated using a split-dosage schedule. Eleven patients suspected of having ovarian carcinoma were injected with 1 mg/kg unlabelled hCTM01, 1 h before receiving 0.1 mg/kg ¹¹¹In-labelled hCTM01 (100 MBq). The amount of radioactivity was determined in resected tumour tissue, various normal tissues and blood samples obtained at laparotomy 6 days postinjection (p.i.). In all patients, the circulating antigen decreased to its nadir after the unlabelled antibody infusion and immune complex formation was demonstrated. Uptake in tumour deposits 6 days p.i. was 11.1 times higher than in normal tissues (P<0.0001) and 5.9 times higher than in blood (P<0.0001). ¹¹¹In activity in liver tissue was comparable to ¹¹¹In uptake in tumour tissue, and considerably lower than previously reported in patients not pretreated with unlabelled antibody. The split-dosing strategy would appear to be advantageous for use of hCTM01 as a specific carrier for the delivery of cytotoxic agents to patients with ovarian cancer. Received: 12 February 1998 / Accepted: 30 April 1998     

Generation of tumor-specific cytotoxic T lymphocytesin vivo by combined treatment with inactivated tumor cells and recombinant interleukin-2

In order to search for a new therapy that would maximize the effect of interleukin-2 (IL-2) in evoking antitumor immunity in vivo, the therapeutic effect of a combination of mitomycin-C(MMC)-treated tumor cells and recombinant IL-2 was examined for its induction of antitumor activity against established melanoma metastasis. In C57BL/6 mice intravenously (i. v.) injected with B16 melanoma cells on day 0, the combined treatment with an intraperitoneal (i. p.) injection of MMC-treated melanoma cells on day 6 and 2500 U rIL-2 (twice daily) on days 7 and 8 markedly reduced the number of pulmonary metastases. This antitumor activity was more effective than that in untreated controls and mice that were injected with MMC-treated melanoma cells alone or rIL-2 alone. When the i. p. injection of MMC-treated tumor cells was replaced by other syngeneic tumor cells, antitumor activity against metastatic melanoma was not induced. The antitumor activity induced by this treatment increased in parallel with an increase in the dose of rIL-2 injected. In contrast, an i. p. injection of soluble tumor-specific antigens alone could induce only a marginal level of antitumor activity, and this activity was not augmented by subsequent i. p. injections of rIL-2. In vivo treatment with anti-CD8 monoclonal antibody (mAb), but not with anti-CD4 mAb or anti-asialo-GM1 antibody, abrogated the antitumor activity induced by this combined therapy. This suggests that the antitumor effect was dependent on CD8⁺ T cells. Lung-infiltrating lymphocytes from mice that had been i. v. injected with melanoma cells 11 days before and were treated with this combined therapy, showed melanoma-specific cytolytic activity. This combined therapy also showed significant antitumor activity against subcutaneously inoculated melanoma cells. These results demonstrate that the combined therapy of an i. p. injection of MMC-treated tumor cells and subsequent and consecutive i. p. administration of rIL-2 increases antitumor activity against established metastatic melanoma by generating tumor-specific CD8⁺ CTL in vivo.     

Effect of local interleukin-2 treatment on spontaneous tumours of different immunogenic strength

Transplantable tumour lines established from spontaneous tumours of BALB/c, CBA, and DBA/2 mice displayed different immunogenic strength. This report describes tumour susceptibility to interleukin-2 (IL-2) therapy in relation to tumour immunogenicity. The following tumour lines were used: X5, X6, and X9 mammary tumours of DBA/2, BALB/c, and CBA origin respectively, X7 carcinoma of BALB/c and X18 papilloma of CBA mice. Two spontaneous tumours of long transplantation history, SL2 lymphoma (SL2) of DBA/2 and Madison lung carcinoma M109 (M109) of BALB/c origin, were used as control systems. Experimental mice were transplanted with different inocula of tumour cells at day 0; treatment with IL-2 was initiated on days 1–3 or delayed until day 10 and consisted of daily injections of low doses of 5000 or 20 000 U/mouse given five times a week for a period of 3 weeks. Treatment of SL2 (2 × 10⁴ cells injected i.p.) consisted of i.p. injections of 5000 or 20 000 U IL-2/mouse given on days 10–14 after tumour transplantation. IL-2 therapy of SL2-bearing DBA/2JIco mice resulted in a significant proportion of cures; however, no response to IL-2 treatment was achieved in SL2-bearing DBA/2CrIiw mice. BALB/c mice with the i.p. transplant of M109 responded to IL-2 treatment with 40% increase in lifespan. The low-dose IL-2 therapy of the five spontaneous tumours resulted, in general, in transient growth inhibition of the i.m. transplants of lines X5, X6, and X7 provided that IL-2 was administered locally. The therapeutic effect depended on the number of transplanted tumour cells, the best results being achieved at cell numbers close to the dose-inducing tumour growth in 50% of animals. We found that the spontaneously arising tumours responding to IL-2 treatment were all slowly growing and immunogenic (X6 and X7) or might have viral association (X5) and, as such, might express foreign antigens. The data suggest a correlation between tumour immunogenicity and the therapeutic effect. However, IL-2 can still exert some effect against tumours with negligible immunogenicity. Received: 16 July 1998 / Accepted: 5 October 1998     

Targeted α-Particle Radiation Therapy of HER1-Positive Disseminated Intraperitoneal Disease: An Investigation of the Human Anti-EGFR Monoclonal Antibody,Panitumumab

Identifying molecular targets and an appropriate targeting vehicle, i.e., monoclonal antibodies (mAb) and their various forms, for radioimmunotherapy (RIT) remains an active area of research. Panitumumab, a fully human and less immunogenic mAb that binds to the epidermal growth factor receptor (Erb1; HER1), was evaluated for targeted α-particle radiation therapy using ²¹²Pb, an in vivo α generator. A single dose of ²¹²Pb-panitumumab administered to athymic mice bearing LS-174T intraperitoneal (i.p.) tumor xenografts was found to have greater therapeutic efficacy when directly compared with ²¹²Pb-trastuzumab, which binds to HER2. A dose escalation study determined a maximum effective working dose of ²¹²Pb-panitumumab to be 20 μCi with a median survival of 35 days versus 25 days for the untreated controls. Pretreatment of tumor-bearing mice with paclitaxel and gemcitabine 24 hours prior to injection of ²¹²Pb-pantiumumab at 10 or 20 μCi resulted in the greatest enhanced therapeutic response at the higher dose with median survivals of 106 versus 192 days, respectively. The greatest therapeutic impact, however, was observed in the animals that were treated with topotecan 24 hours prior to RIT and then again 24 hours after RIT; the best response from this combination was also obtained with the lower 10-μCi dose of ²¹²Pb-panitumumab (median survival >280 days). In summary, ²¹²Pb-panitumumab is an excellent candidate for the treatment of HER1-positive disseminated i.p. disease. Furthermore, the potentiation of the therapeutic impact of ²¹²Pb-pantiumumab by chemotherapeutics confirms and validates the importance of developing a multimodal therapy regimen.