The distribution of the therapeutic monoclonal antibodies cetuximab and trastuzumab within solid tumors

Background  

Poor distribution of some anticancer drugs in solid tumors may limit their anti-tumor activity.     

Relationship between antigen density and immunotherapeutic response elicited by monoclonal antibodies against solid tumors

The relationship between the level of cell surface antigen expression and solid tumor immunotherapy with monoclonal antibody (MoAb) was evaluated. Two MoAb's that were shown effective in the passive therapy of breast carcinomas of human origin, established and growing in female Swiss nude mice, were used for these studies. Several groups of tumors were produced from cell cultures of different passages; each cell culture possessed a distinct target antigen level. Results from immunotherapy experiments demonstrated that the amount of tumor reduction response after MoAb therapy was proportional to the antigen density at the cell surface. Analysis of these data indicated a theoretical improbability of a single MoAb treatment being able to completely eradicate solid tumors and may necessitate the use of multiple MoAb's to circumvent this problem.     

Monoclonal antibodies in therapy of solid tumors

Monoclonal antibodies have become increasingly used therapeutic agents for the treatment of solid cancer. Many are now being tested as components of adjuvant or first-line therapies to assess their efficacy in improving or prolonging survival. Selected unconjugated antibodies can exert clinically significant antitumor effects in many cancers. Antibody conjugates have been used to deliver toxic principles, such as radioactive particles, chemotherapeutic agents, and catalytic toxins, with increasing success in clinical trials.     

Dynamic interaction of 111indium-labeled monoclonal antibodies with surface antigens of solid tumors visualized in vivo by external scintigraphy

Two 111indium-labeled murine monoclonal antibodies (MoAb), D3 and 9.2.27, directed to tumor antigens of L-10 hepatocarcinoma and human melanoma, respectively, selectively localized antigen-positive target cells in guinea pigs and nude mice. The fate of MoAb differed in the two antigen-antibody systems after reacting with their corresponding tumor antigens in vivo as reflected by patterns of distribution and turnover in vivo. The 9.2.27 localized in melanoma xenograft in nude mice after intravenous administration with slow loss from tumor but more rapid loss from normal tissues and thus demonstrated optimal imaging of small tumors (approximately equal to 5 mm) between 3 and 6 days after injection of the radiolabeled antibody. In contrast, D3 demonstrated a biphasic localization in guinea pig L-10 hepatocarcinoma with a maximal activity on the 2d day after administration and showed rapid loss from both tumor and normal tissues. Nonspecific localization of antibodies in liver and in kidney was found both in syngeneic (nude mice) and xenogeneic (guinea pig) hosts but was more pronounced in the xenogeneic species. These results indicate that the nature of the antigen-antibody interaction may be of importance in selecting MoAb for both diagnosis and therapy of malignant diseases.     

特异性抗膀胱癌单克隆抗体对膀胱肿瘤的临床研究

 采用一组特异性抗膀胱癌单克隆抗体MAb（T₁₆、M₃₄₄、T₁₃₈、T₄₃），以APAAP免疫细胞化学方法，对63例膀胱移行细胞癌及10例非肿瘤患者的膀胱冲洗液作了研究。T₁₆在全部63份标本呈阳性。随着肿瘤分期、分级的升高，M₃₄₄阳性率逐渐降低，T₁₃₈及T₄₃阳性率逐渐升高。此三种MAb与肿瘤复发也有一定的对应关系。尿细胞学与MAb联合应用，可弥补相互间的不足，使肿瘤检出率进一步提高。结果表明：M₃₄₄、T₁₃₈及T₄₃MAb的阳性表达与肿瘤的分期、分级及预后密切相关，可作为膀胱肿瘤恶性程度、复发及早期诊断的估价指标。     

Active specific immunotherapy with antiidiotypic antibodies in patients with solid tumors

Tumor-associated antigens (TAA) provide appropriate targets for selective manipulation of the patient's immune response in the immunotherapy of cancer. Active specific immunotherapy utilizing antiidiotypic antibodies to anti-TAA antibodies has been implemented in phase I clinical trials both in patients with colorectal carcinoma and in those with melanoma. The theoretical basis for immunotherapy with antiidiotypic antibodies, the results of these clinical trials, and an evaluation of appropriate parameters for future clinical trials of active specific immunotherapy with antiidiotypic antibodies in patients with solid tumors are reviewed.     

Recombinant-human-erythropoietin (rhuepo) in the treatment of anemia associated with solid tumors with or without bone-marrow infiltration

This study was designed to test the efficacy of rHuEPO in inducing an increase of at least 2 gr/dl over baseline hemoglobin levels in patients affected by solid tumors, with or without bone marrow invasion. The treatment plan consisted of the administration of rHuEPO 150 U/kg of body weight 3 times/week for 6 weeks. In responding patients, a maintenance schedule of 150 U/kg/week was given for a further 6 weeks. Twenty patients with hemoglobin levels of between 8 and 10 gr/dl were treated, 10 of whom (50%) had bone marrow infiltration. There was a significant difference between median baseline serum EPO levels in patients with and without bone marrow invasion (123.5 vs 40 mU/ml, p=0.002). A response was achieved in 10 of the 20 cases (50%), the median duration being 14 weeks (range 3-34+). Three of the 10 patients with bone marrow involvement responded to treatment, as opposed to 7 without bone marrow invasion (30% vs 70%; p=0.179). The treatment was well tolerated and did not give rise to any severe side effects, These data suggest that the rHuEPO 150 U/kg 3 times/wk controls chronic anemia in 50% of patients affected by solid tumors. Good efficacy of rHuEPO treatment was observed in patients without bone marrow infiltration.     

Local gene therapy of solid tumors with GM-CSF and B7-1 eradicates both treated and distal tumors

Gene therapy-induced expression of immunostimulatory molecules at tumor cell level may evoke antitumor immune mechanisms by recruiting and enhancing viability of antigen-processing cells and specific tumoricidal lymphocytes. The antitumor efficacy of a plasmid, coding for granulocyte-macrophage colony-stimulating factor (GM-CSF) and the B7-1 costimulatory immune molecule, delivered into growing solid tumors by electroporation was investigated. Murine fibrosarcomas (JBS) growing in Balb/C mice (相似文献   

Micropharmacology of monoclonal antibodies in solid tumors: direct experimental evidence for a binding site barrier.

Monoclonal antibodies (MAbs) often distribute nonuniformly in tumors. In part, that observation reflects intrinsic heterogeneity within the tumor; in part, it reflects poor penetration through tumor substance. Several years ago, we proposed the "binding site barrier" hypothesis (J.N. Weinstein, R.R. Eger, D.G. Covell, C.D.V. Black, J. Mulshine, J.A. Carrasquillo, S.M. Larson, and A.M. Keenan, Ann. NY Acad. Sci., 507: 199-210, 1987; K. Fujimori, D.C. Covell, J.E. Fletcher, and J.N. Weinstein, Cancer Res., 49: 5656-5663, 1989), the idea that antibodies (and other ligands) could be prevented from penetrating tumors by the very fact of their successful binding to target antigen. Calculations suggested that this might be a significant factor in the therapy of even microscopic nodules. The higher the affinity and the higher the antigen density, the greater the barrier. Here, we provide direct experimental evidence of such a barrier to the percolation of D3 MAb through intradermally implanted line 10 carcinoma of guinea pigs. After affinity purification using glutaraldehyde-fixed line 10 cells, the D3 had an average immunoreactivity of 88%, a binding constant of 1.6 +/- 0.3 (SEM) x 10(10) M-1, and saturation binding of 355,000 +/- 15,000 molecules/cell. Using a combination of double-label autoradiography and double-chromagen immunohistochemistry, we determined simultaneously the distribution of (a) i.v. injected D3 MAb; (b) coinjected isotype-matched control IgG (BL3); (c) D3 antigen; (d) blood vessels. The previously developed mathematical models aided in the design of these experiments. Double immunochemical staining of the tumors showed antigen-rich patches 100-800 microns across, surrounded by blood vessels. At a low MAb dose (30 micrograms), binding to antigen severely hindered penetration into antigenic patches as small as 200 microns, even at 72 h. Explanation of this finding by a physical barrier was ruled out by the observation that BL3 distributed uniformly in the same patches. At a higher dose (1000 micrograms), the binding site barrier could be partially overcome. The same general principles of micropharmacology may apply to biological ligands other than antibodies, including those secreted by genetically modified cells.     

Monoclonal antibodies to a rat colon carcinoma: model for monoclonal antibody therapy of solid tumors

Tumor progression, lung metastasis, and death occur in tumor-bearing BD IX syngeneic rats in a fashion similar to the course of patients with metastatic colon cancer. In an effort to establish a relevant model for monoclonal antibody (MoAb) therapy of tumors, we generated murine MoAb against DHD/TR, a dimethylhydrazine-induced rat colon carcinoma which has been adapted to cell culture. Murine MoAb 17B10 E4 (E4) reacts with the TR tumor and shows weak immunoperoxidase reactivity with normal rat tissues. Murine MoAb 5F7 D3 (D3) reacts with the tumor and a variety of normal rat epithelia. Both are IgG2a and mediate cytotoxicity by rat peripheral blood mononuclear cells. 18D5 F6 (F6) also reacts with the tumor and normal tissues but is an IgG2b and does not mediate cytotoxicity in the presence of rat effector cells. Iodinated E4 and D3 antibodies retained their immunoreactivity. E4 revealed 9.8 x 10(5) antigenic sites per TR cell, with an affinity constant of 9.35 x 10(7) M-1, while D3 demonstrated 2.5 x 10(6) antigenic sites and an affinity constant of 4.2 x 10(7) M-1. Immunoblotting showed that the antigens recognized by D3 and E4 are glycoproteins with molecular weights of 27,000 and 66,000, respectively. F6 failed to react with its antigen present in the blot. This rat colon carcinoma and the monoclonal antibodies described here may provide experimental data useful for implementing monoclonal antibodies in cancer therapy.     

Antiangiogenic radioimmunotherapy of human solid tumors in SCID mice using (125)I-labeled anti-endoglin monoclonal antibodies.

Endoglin (CD105), which is a component of the TGF-beta receptor complex, is highly expressed at the surface of proliferating human endothelial cells such as those of tumor vessels. In the present study, we tested the antitumor efficacy of (125)I-labeled anti-endoglin monoclonal antibodies (MAbs), SN6f and SN6j, against s. c. tumors of MCF-7 human breast cancer cells in SCID mice by i.v. administration. SN6f and SN6j cross-react weakly with mouse endothelial cells, but show no significant reactivity with MCF-7 tumor cells. These MAbs are effectively internalized into the cells after binding to the cell surface antigen of endothelial cells. Four groups of SCID mice (n = 10 or 9 in each group) inoculated s.c. with 8 x 10(6) MCF-7 cells were treated with (125)I-SN6f (10 microCi), (125)I-SN6j (10 microCi), a (125)I-labeled isotype-matched control IgG (10 microCi) or PBS. The systemic therapy was performed in 2 series, i.e., on days 3, 5, 7 and days 58, 60, 62. Both (125)I-SN6f and (125)I-SN6j showed significant growth suppression of the tumors, whereas the (125)I-labeled control IgG did not show any significant antitumor efficacy. No significant toxicity or weight loss was observed in mice treated with either (125)I-SN6f or (125)I-SN6j. After 100 days of observation, autopsies revealed no significant organ damage. Our results show the possible usefulness of antiangiogenic radioimmunotherapy using (125)I-labeled anti-endoglin MAbs.     

Immunocytochemical study with monoclonal antibodies to progesterone receptor in human breast tumors

Mouse hybridomas secreting monoclonal antibodies against rabbit uterine progesterone receptor (PR) have been prepared. Several of these immunoglobulins exhibited high affinity towards human progesterone receptor and two (LET 126 and LET 64) were selected as giving the best immunoperoxidase staining of human progesterone target organs. Using the indirect peroxidase-antiperoxidase method of Sternberger, optimal conditions for demonstrating PR involved brief fixation of frozen sections with formaldehyde-containing fixatives, among them picric acid-paraformaldehyde. This method allowed us to detect the receptor in breast carcinoma epithelial cells, T47D cell line, and uterine endometrium and myometrium. No staining was observed in intestine and muscle. Specific staining for PR was confined to the nucleus, irrespective of the concentration of progesterone in the blood of the patient. In a preliminary study of 27 human breast cancers by the immunocytochemical method, the presence or absence of nuclear staining for PR correlated well with the concentration of cytosolic progesterone receptor determined by a steroid-binding assay on tumor extracts. Differences in the intensity and distribution of staining within a section were observed, suggesting heterogeneity of the PR content of breast cancer cells. In 19 tumors, the immunocytochemical method for PR localization was also used in combination with a slightly modified Abbott ER-ICA staining for estrogen receptor to compare the distribution of both receptors within the same biopsy on adjacent frozen sections. Various combinations of estrogen receptor and PR contents that have been determined by steroid-binding assay have also been detected by the double immunocytochemical assay.     

Strategies to enhance monoclonal antibody uptake and distribution in solid tumors

Despite the significant resources dedicated to the development of monoclonal antibody (mAb) therapies for solid tumors, the clinical success, thus far, has been modest. Limited efficacy of mAb in solid tumors likely relates to unique aspects of tumor physiology. Solid tumors have an aberrant vasculature and a dense extracellular matrix that slow both the convective and diffusive transport of mAbs into and within tumors. For mAbs that are directed against cellular antigens, high antigen expression and rapid antigen turnover can result in perivascular cells binding to and eliminating a significant amount of extravasated mAb, limiting mAb distribution to portions of the tumor that are distant from functional vessels. Many preclinical investigations have reported strategies to improve mAb uptake and distribution; however, to our knowledge, none have translated into the clinic. Here, we provide an overview of several barriers in solid tumors that limit mAb uptake and distribution and discuss approaches that have been utilized to overcome these barriers in preclinical studies.     

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.     

Intraperitoneal radio-localization of tumors pre-targeted by biotinylated monoclonal antibodies

We describe 2-step and 3-step strategies for intraperitoneal tumor radio-localization by means of monoclonal antibodies (MAbs). Nude mice bearing intraperitoneal human colon carcinoma tumors were injected i.p. with biotinylated MAb AUAI, followed 24 hr later by radioiodinated streptavidin (2-step). The uptake of radioactivity in tumor and normal tissues was measured 4 hr after injection of radioactive compound. A 3-step strategy consisted in administering biotinylated antibody, cold avidin after 24 hr and 111In-labelled biotin after a further 4 hr; mice were then killed 2 hr later. Tumor localization of intraperitoneally-administered biotinylated antibody and direct targeting of radioactive streptavidin to biotinylated antibody bound to tumor sites were demonstrated using immunohistochemistry and autoradiography. Our results show that (i) the 2-step approach increased the percentage of radioactivity uptake by tumor with respect to directly labelled antibodies (24% vs. 6%) and improved the tumor/non-tumor ratio; (ii) the 3-step approach allowed faster blood clearance of the radioactive probe (111In-biotin) and yielded high tumor/non-tumor ratios. "Pre-targeting" methods appear to have advantages over the conventional 1-step approach with directly radiolabelled antibody.     

Anti-tumoral effect of desmethylclomipramine in lung cancer stem cells

Lung cancer is the most feared of all cancers because of its heterogeneity and resistance to available treatments. Cancer stem cells (CSCs) are the cell population responsible for lung cancer chemoresistance and are a very good model for testing new targeted therapies. Clomipramine is an FDA-approved antidepressant drug, able to inhibit in vitro the E3 ubiquitin ligase Itch and potentiate the pro-apoptotic effects of DNA damaging induced agents in several cancer cell lines. Here, we investigated the potential therapeutic effect of desmethylclomipramine (DCMI), the active metabolite of Clomipramine, on the CSCs homeostasis. We show that DCMI inhibits lung CSCs growth, decreases their stemness potential and increases the cytotoxic effect of conventional chemotherapeutic drugs. Being DCMI an inhibitor of the E3 ubiquitin ligase Itch, we also verified the effect of Itch deregulation on CSCs survival. We found that the siRNA-mediated depletion of Itch induces similar anti-proliferative effects on lung CSCs, suggesting that DCMI might exert its effect, at least in part, by inhibiting Itch. Notably, Itch expression is a negative prognostic factor in two primary lung tumors datasets, supporting the potential clinical relevance of Itch inhibition to circumvent drug resistance in the treatment of lung cancer.     

Generation of monoclonal antibodies reactive with nuclear proteins of human primary breast tumors

Nuclear proteins were extracted from purified nuclei of human primary breast tumors (BrT) and bladder tumors and of human normal breast, kidney and lymphocytes by enzymatic treatment. SDS-Polyacrylamide gel electrophoresis of nuclear proteins from breast tumors showed different bands in the molecular weight zones from 25 to 220 kDa which were absent or present only as traces in normal breast tissue. Murine monoclonal antibodies (MAbs) have been produced using nuclear extracts of human primary breast tumors as immunogens. Approximately 2,000 hybridomas were generated from 5 hybridizations. According to their reactivity to BrT nuclear extracts and mammary carcinoma cell line MCF-7, seven hybridomas were selected and cloned. They were further characterized with histological immunoperoxidase assays of formaldehyde-fixed BrT paraffin tissue sections. MAb 6A3 particularly gave strong nuclear staining with all BrT specimens while MAb 1D8 showed both nuclear and cytoplasmic staining with only some of them. Specimens from mammoplasty did not react with these MAbs. Immunoblotting of BrT nuclear extracts as developed with MAbs 6A3 and 1D8 revealed major protein bands with molecular weight of 120 and 130 kDa. The potential use of these MAb-defined BrT-related nuclear proteins as markers for human breast cancer was suggested.     

Treatment of extra-abdominal desmoid tumors with interferon-alpha with or without tretinoin

BACKGROUND AND OBJECTIVES: Surgery is the main treatment for extra-abdominal desmoid tumors, but the results of further management remain uncertain. Therefore, a retrospective analysis was undertaken to evaluate the toxicity and efficacy of treatment with interferon-alpha (IFN-alpha) +/- tretinoin in this setting. METHODS: Thirteen patients with extra-abdominal desmoid tumors and a median age of 32 years (range, 15-73) received IFN-alpha. Seven of these patients received a combination of IFN-alpha and tretinoin in order to test further enhancement. RESULTS: After a mean observation period of 27 +/- 15 months (mean +/- standard deviation) under treatment with IFN-alpha +/- tretinoin, local control was seen in 11 of 13 patients (85%). Seven patients had no evidence of disease at a mean disease-free interval of 22 +/- 18 months; in two patients progressive disease occurred after only 7 and 9 months, respectively, of observation. In another four patients, progression of the desmoid tumor was stabilized. CONCLUSIONS: The data of this retrospective, nonrandomized study on therapy with IFN-alpha +/- tretinoin suggest that such treatment may be effective in prolonging the disease-free interval of patients after intralesional or marginal surgery. Because of the encouraging response rate, this regimen appears to be another nonsurgical treatment alternative.