A quantitative analysis of tumor specific monoclonal antibody uptake by human melanoma xenografts: effects of antibody immunological properties and tumor antigen expression levels.

The time-dependent (5 min-72 h) localization of 3 radiolabeled anti-melanoma monoclonal antibodies (MAbs 436, IND1, and 9.2.27) was studied in paired label experiments in small (4-12 mg) s.c. human melanoma xenografts (SK-MEL-2 and M21) in athymic nude mice. MAb 436 recognizes a Mr 125,000 cell surface melanoma-associated glycoprotein antigen (125 kDa-MAA); MAbs IND1 and 9.2.27 recognize a high molecular weight melanoma-associated antigen, but with equilibrium association constants differing by 2 orders of magnitude (10(8)-10(10) M-1). The two tumors were found to differ in their antigen expression levels and in both interstitial and vascular volumes. Accumulation of MAbs in both tumors was determined primarily by antigen expression levels and also by physiological factors such as vascular permeability and vascular volume; at the dose administered (20 micrograms/mouse), differences in MAb affinity among specific MAbs had minimal effect on accumulation. Quantitative flow cytometry measurements showed that antigen expression in vivo differed from that of cultured tumor cells. In vivo, expression of the Mr 125,000 MAA decreased by a factor of about 2.5 in both tumors. In contrast, the in vivo expression of the high molecular weight MAA decreased in M21 tumors but increased by 2.0-3.5-fold in SK-MEL-2 tumors. Data were analyzed using a three-compartment pharmacokinetic model (C. Sung et al., Cancer Res., 52:377-384, 1992) to provide plasma-to-tissue transport constants (k), the interstitial fluid flow rate (L), and estimates of the in vivo interstitial MAb binding site concentration (B0). For all MAbs, the plasma-to-tissue transport constants were consistently greater for M21 tumors (0.44-0.85 microliter/min/g) than for SK-MEL-2 tumors (0.28-0.66 microliter/min/g), and values of k for both tumors were approximately 1 order of magnitude greater than those for skeletal muscle (0.06-0.08 microliter/min/g). The model-estimated binding site concentration of melanoma-specific antibodies was 15-70 times lower than that predicted by experimental measurements of tumor antigen concentrations. Factors that may contribute to this discrepancy include inaccessibility of tumor cell binding sites to MAb and MAb catabolism. In summary, these results indicate that, for the MAb dose used in this study, variables pertaining to the tumor target (i.e., antigen expression levels, vascular volume, and vascular permeability) are the most important for determining MAb accumulation in tumors.     

Molecular cloning and characterization of antigens expressed on rat tumor vascular endothelial cells

We have previously prepared monoclonal antibodies (MAbs) against tumor vasculature using cultured rat tumor endothelial cells (TECs) isolated from solid KMT-17 tumors and identified 40 and 80 kDa antigens recognized by TES-17 and TES-23 MAbs, respectively. To clarify the nature of antigens on tumor vasculature, molecular cloning was conducted by screening the rat TEC cDNA library. Two antigens were detected: a 40 kDa OTS-8 antigen, which had been defined as a differentiation marker for osteoblastic lineage, and an 80 kDa CD44H antigen. Northern blots showed that OTS-8 mRNA was expressed exclusively in the lung, in addition to TECs, while CD44H mRNA was detected in the lung, intestine, spleen, thymus and peripheral blood cells, in addition to TECs. Immuno-histochemistry of KMT-17 tumors revealed that OTS-8 and CD44 were expressed on sprouting TECs. In addition, TES-23 MAb stained TECs of tubular vessels as well as sprouting TECs, but anti-rat CD44 MAbs stained only sprouting TECs. PCR showed that CD44 cDNA with a splice in exon 6 (CD44ex6) was present in rat TECs at low levels. Our results indicate that OTS-8 and CD44 are expressed on rat sprouting TECs and that TES-23 MAb recognizes CD44H on sprouting TECs as well as an unknown epitope on TECs of tubular vessels that could not be recognized by anti-rat CD44 MAbs.     

Rat monoclonal antibodies produced against rat colorectal adenocarcinomas define tumor- and colon-associated, auto-immunogenic antigens.

Monoclonal antibodies (MAbs) were derived from rats immunized against allo- and syngeneic rat colon carcinomas. Screening was performed by immunohistochemistry modified for MAbs of the same species as the tissue used for frozen sections. From 2 fusions, several MAbs were found that bound to syngeneic tumor tissue but showed little or no staining of normal adult tissues. Another group of MAbs demonstrated an intracellular staining of tumor cells and staining of mucus and goblet cells in the distal 2/3 of the normal colon. A third group of MAbs showed staining of both tumor and normal colon tissue. Staining patterns were reproduced with 6 MAbs selected from the first 2 groups after purification and biotinylation. One MAb, 10B12, recognized an antigen expressed in 10/11 colon carcinomas, the lowest parts of colonic crypts, intracellularly in a subpopulation of pancreatic acinar cells and mucus in antrum. It was used for affinity purification of a high-molecular-weight antigen, to which 3 of the other rat MAbs also bound. This antigen was also bound by antibodies to blood group A and isogloboseries carbohydrate determinants. Competition with the labelled 10B12 MAb for binding to the purified antigen was demonstrated in sera of tumor-bearing and immune rats. Thus, this high-molecular-weight glycoprotein expressed both auto-antigenic, tumor-associated and tissue-type-restricted epitopes. The 10B12 MAb homogeneously stained the majority of colorectal carcinomas and the antigenic determinant was expressed on the cell surface of 12/13 tissue-cultured colon-carcinoma clones. Modulation of the cell-surface antigen phenotype by recombinant rat interferon-gamma markedly increased expression of this determinant.     

Enhanced tumor uptake of macromolecules induced by a novel vasoactive interleukin 2 immunoconjugate

Low uptake of monoclonal antibodies (MAbs) in cancer lesions is a significant problem in cancer therapy. Recent studies have shown that antibody uptake in tumor is controlled in large part by the tumor blood flow and the vascular permeability of the tumor endothelium. We have hypothesized that these physiological properties of tumor vessels may be altered by pretreatment with vasoactive drugs or peptides linked to tumor-specific MAbs. To test this hypothesis, two MAbs, Lym-1 directed against human malignant lymphomas and B72.3 reactive with the TAG-72 antigen expressed in solid tumors, were chemically conjugated with human recombinant interleukin 2 (IL-2). IL-2 has been used in humans to activate lymphokine-activated killer cells for the treatment of cancer but is also known to produce a generalized vascular permeability by an unknown mechanism when used systemically. Chemical conjugation of IL-2 to MAbs appears to destroy its cytokine function as shown by T-cell proliferation studies in vitro. Despite this finding, MAb/IL-2 immunoconjugates retain their ability to produce an enhanced vascular permeability when injected i.v. into nude mice bearing relevant tumor models only. Biodistribution studies using 125I-labeled tracer Lym-1 have demonstrated that the Lym-1/IL-2 immunoconjugate can increase antibody uptake in tumor by a factor of 4 in a time (2.5-h pretreatment)- and dose (30 micrograms/mouse)-dependent manner. In contrast, treatment of mice with free IL-2 and antibody showed this effect in all organs of the mouse including the tumor. Bidirectional crossover imaging studies in individual tumor-bearing nude mice showed improved uptake and decreased blood pool when the MAb/IL-2 immunoconjugates were used compared to controls. Finally, tumor blood flow and vascular permeability studies demonstrate that the physiological effect of the MAb/IL-2 is due to a reversible and specific vascular leakage at the tumor site. These studies indicate that pretreatment with this novel immunoconjugate may enhance the diagnostic and therapeutic potential of MAbs, drugs, and other macromolecules for the treatment of cancer.     

Fetal antigen 2 in primary and secondary brain tumors.

Immunohistochemical deposition and distribution of fetal antigen 2 (FA2) was examined in normal brain tissue and in primary and metastatic tumors of the brain. In normal brain tissue FA2 was exclusively found linearly around the vessels, along pia and in arachnoidea. A similar localization was seen in primary brain tumors except in gliosarcoma where FA2 was distributed diffusely in the sarcoma region and was absent in the glioma region. In metastatic carcinoma with tumor stroma a diffuse staining reaction was seen in the stroma and with a basement membrane (BM) like staining at the tumor cell/stroma interface. Intracytoplasmic FA2 staining of the tumor cells was seen in areas without tumor stroma. In metastatic melanoma a BM like FA2 staining was seen around and between individual tumor cells. The staining patterns seen in the metastatic tumors were in accordance with that of the corresponding primary tumors.     

Effect of inhibition of vascular endothelial growth factor signaling on distribution of extravasated antibodies in tumors

Antibodies and other macromolecular therapeutics can gain access to tumor cells via leaky tumor vessels. Inhibition of vascular endothelial growth factor (VEGF) signaling can reduce the vascularity of tumors and leakiness of surviving vessels, but little is known about how these changes affect the distribution of antibodies within tumors. We addressed this issue by examining the distribution of extravasated antibodies in islet cell tumors of RIP-Tag2 transgenic mice and implanted Lewis lung carcinomas using fluorescence and confocal microscopic imaging. Extravasated nonspecific immunoglobulin G (IgG) and antibodies to fibrin or E-cadherin accumulated in irregular patchy regions of stroma. Fibrin also accumulated in these regions. Anti-E-cadherin antibody, which targets epitopes on tumor cells of RIP-Tag2 adenomas, was the only antibody to achieve detectable levels within tumor cell clusters at 6 hours after i.v. injection. Treatment for 7 days with AG-013736, a potent inhibitor of VEGF signaling, reduced the tumor vascularity by 86%. The overall area density of extravasated IgG/antibodies decreased after treatment but the change was less than the reduction in vascularity and actually increased when expressed per surviving tumor vessel. Accumulation of anti-E-cadherin antibody in tumor cell clusters was similarly affected. The patchy pattern of antibodies in stroma after treatment qualitatively resembled untreated tumors and surprisingly coincided with sleeves of basement membrane left behind after pruning of tumor vessels. Together, the findings suggest that antibody transport increases from surviving tumor vessels after normalization by inhibition of VEGF signaling. Basement membrane sleeves may facilitate this transport. Antibodies preferentially distribute to tumor stroma but also accumulate on tumor cells if binding sites are accessible.     

Immunohistochemical determination of inducibility phenotype with a monoclonal antibody to a methylcholanthrene-inducible isozyme of cytochrome P-450

A monoclonal antibody (MAb) to a methylcholanthrene (MC)-induced cytochrome P-450, designated MAb 1-7-1, was used for immunohistochemical staining of formalin-fixed tissues from oil- and MC-treated C57BL/6, DBA/2, and [(C57BL/6 X DBA/2) F1 X DBA/2] F2 mice. An avidin-biotin-peroxidase complex immunohistochemical technique was used. For controls, the tissues were also exposed to MAbs 1-48-5 and HyHel-9 (to egg white lysozyme). In liver, MAb 1-7-1 specifically stained the cytoplasm of centrilobular hepatocytes of C57BL/6 mice treated with MC (80 mg/kg) 48 h before kill; staining was not observed with vehicle-treated C57BL/6 mice, with oil- or MC-treated DBA/2 mice, or with comparable antibody concentrations of control MAbs 1-48-5 or HyHel-9. In the F2 mice, about 50% were expected to be MC inducible (AhbAhd). Inducibility phenotype was determined by measuring the conversion of [14C]MC to oxidized and conjugated products by liver homogenates. In freshly fixed material from MC-treated mice, those livers shown by the determination of phenotype to be inducible also stained with MAb 1-7-1, whereas those not induced were immunohistochemically negative. Furthermore, there was a significant positive correlation between degree of staining and the level of MC-metabolizing activity measured biochemically. The immunohistochemical procedure was also accurate in determination of inducibility phenotype of livers that had been in paraffin blocks for up to 2 yr if more concentrated antibody was used. In lung, MAb 1-7-1 stained specifically the alveolar walls and endothelium of blood vessels in MC-induced C57BL/6 mice only; the control MAbs and other mice gave negative results. Similarly, in kidney MAb 1-7-1 stained only glomeruli and interstitial tissue of MC-induced C57BL/6 mice and only endothelium of blood vessels in the colons of these mice. These observations are consistent with induction of the cytochrome P-450 recognized by MAb 1-7-1 in the endothelial cells of extrahepatic tissue. Immunohistochemical staining with MAb thus shows great promise for highly specific localization of particular species of cytochromes P-450 in tissues, for in situ quantification of these enzymes, and for determination of inducibility phenotype with fixed material.     

Distinguishing malignant mesothelioma from pulmonary adenocarcinoma: an immuno-histochemical approach using a panel of monoclonal antibodies

A panel of six monoclonal antibodies (MAbs) was employed to evaluate antigen expression in pulmonary adenocarcinomas and mesotheliomas. Monoclonal anti-human milk fat globulin (HMFG-2), anti-carcinoembryonic antigen (NP-2), anti-epithelial membrane antigen (EMA), anti-cytokeratin (PKK-1), anti-tumor-associated antigen 72 (B72.3), and anti-human myelomonocytic antigen (Leu M-1) antibodies were used to localize their respective antigens in formalin-fixed, paraffin-embedded tumors by using the avidin-biotin-complex immunoperoxidase technique. In all, 28 mesotheliomas obtained from Ohio State University Anatomic Pathology files and from a Southwest Oncology Group (SWOG) protocol were compared to 22 pulmonary adenocarcinomas by using this MAb panel. None of the mesotheliomas demonstrated positive staining with MAbs NP-2 (anti-CEA) or Leu M-1. However, 95% (21/22) of adenocarcinomas stained with one of these two antibodies. Although neither of these two MAbs stained all adenocarcinomas, each antibody demonstrated positive immunostaining in more than 90% of the adenocarcinomas studied. Therefore, MABs NP-2 and Leu M-1 are, individually, quite useful for distinguishing mesothelioma from adenocarcinoma. However, in our study, no single MAb could be used to distinguish these two tumor types in every case. MAb B72.3 stained 91% (20/21) adenocarcinomas but also stained 7% (2/28) of mesotheliomas. MAb HMFG-2 reacted positively with 95% of adenocarcinomas, but also stained 39% of the mesotheliomas, usually in a membranous pattern. MAbs EMA and PKK-1 were not found useful in distinguishing mesothelioma from adenocarcinoma. We conclude that MAbs Leu M-1 and NP-2 were both useful in distinguishing mesothelioma from pulmonary adenocarcinoma in that positive staining was demonstrated in adenocarcinomas and not mesotheliomas.     

Predicted and observed effects of antibody affinity and antigen density on monoclonal antibody uptake in solid tumors.

The uptake and binding of monoclonal antibodies (MAbs) in solid tumors after a bolus i.v. injection are described using a compartmental pharmacokinetic model. The model assumes that MAb permeates into tumor unidirectionally from plasma across capillaries and clears from tumor by interstitial fluid flow and that interstitial antibody-antigen interactions are characterized by the Langmuir isotherm for reversible, saturable binding. Typical values for plasma clearance and tumor capillary permeability of a MAb and for interstitial fluid flow and interstitial volume fraction of a solid tumor were used to simulate the uptake of MAbs at various values of the binding affinity or antigen density for a range of MAb doses. The model indicates that at low doses, an increase in binding affinity may lead to an increase in MAb uptake. On the other hand, at doses approaching saturation of antigen or when uptake is permeation limited, an increase in the binding affinity from moderate to high affinity will have only a small effect on increasing MAb uptake. The model also predicts that an increase in antigen density will greatly increase MAb uptake when uptake is not permeation limited. Our experiments on MAb uptake in melanoma tumors in athymic mice after injection of 20 micrograms MAb (initial plasma concentration, about 120 nM) are consistent with these model-based conclusions. Two MAbs differing in affinity by more than 2 orders of magnitude (3.8 x 10(8) M-1 and 5 x 10(10) M-1) but with similar in vivo antigen densities in M21 melanoma attained similar concentrations in the tumor. Two MAbs of similar affinity but having a 3-fold difference in in vivo antigen density in SK-MEL-2 melanoma showed that the MAb targeted to the more highly expressed antigen attained a higher MAb concentration. We also discuss the model predictions in relation to other experiments reported in the literature. The theoretical and experimental findings suggest that, for high dose applications, efforts to increase MAb uptake in a tumor should emphasize the identification of an abundantly expressed antigen on tumor cells more than the selection of a very high affinity MAb.     

Monoclonal antibody against a tumor-associated sialoglycoprotein of superficial papillary bladder tumors and cervical condylomas.

A mouse IgG1 monoclonal antibody (MAb), 19A211, defining a tumor-associated cell-surface antigen of superficial papillary bladder tumors, was generated by immunizing with fresh bladder tumor cells mice neonatally injected with normal human urothelial cells. The reactivity of the antibody with cell lines was examined by indirect immunofluorescence staining and was restricted to 3/14 bladder cancer lines and 3/31 cancer cell lines of non-bladder origin, including HeLa cervical cancer. No normal fibroblast, kidney cells, EBV-lymphocytes, erythrocytes or leukocytes expressed the antigen. Reactivity of MAb 19A211 was well preserved on tissue paraffin sections. Immunoperoxidase staining of normal adult or fetal tissues showed no reactivity except for a patchy or uniform staining of umbrella cells in 6/23 adult and 1/4 fetal urothelium samples. Positive and often heterogeneous staining was observed on 24/38 papillary superficial tumors (Ta) and 4/5 carcinoma in situ bladder lesions but on only 4/20 infiltrating tumors. It was also observed on 5/6 cervical condylomas and one bladder condyloma, but none of 6 penile or vulvar condylomas. All other tumors tested were negative. The antigenic determinant is present on a heterogeneous group of proteins with molecular weights ranging from 90 to 200 kDa. It is sensitive to periodate treatment and to neuraminidase but only partially sensitive to proteases. MAb 19A211 is different from other reported MAbs with similar reactivity to superficial bladder tumors and umbrella cells of normal urothelium. When tested in competition assays, several of these MAbs, but not 19A211, were found to react with Lewis X blood group determinant. Our results suggest that 19A211 may be useful for detection and stratification of bladder tumors.     

Lack of specificity of endoglin expression for tumor blood vessels.

A monoclonal antibody (MAb; A11) has been raised following mouse immunization with cultured human microvascular endothelial cells. The MAb showed a strong positivity within tumor vessels in glioblastoma and breast carcinoma samples, and the distribution was consistent with antigen association with vascular endothelial cells. A purification procedure of the antigen was developed starting from DG-RSV-LT-2, an immortalized human endothelial cell line. Molecular mass, N-terminal sequence of the purified antigen and localization on endothelial cell surface allowed identification with human endoglin (CD105). Flow cytometry analysis of a group of normal and transformed cell lines showed that, besides endothelial cells and myelocytic leukemia cells already shown to be positive, fetal fibroblasts, choriocarcinoma, fibrosarcoma and rhabdomyosarcoma cell lines were also positive for this antigen. Immunohistochemic analysis of several normal adult tissues revealed a more extensive presence of the antigen in normal vessels compared to that described with previously characterized antibodies. In fact, even though the staining was weaker than in tumor tissues, all tissues were found to be positive, at least in microvessels, except for normal breast. Moreover, in some tissues (glands and reproductive tract) a positive reaction was observed in the stroma. Since endoglin has been proposed as a possible target for antiangiogenic therapy in tumor patients and our data demonstrate a sizable amount of endoglin in normal vessels and stroma, its clinical use should be carefully reevaluated.     

A rapid and efficient in vivo method for determining the biologic efficacy of monoclonal antibodies in animal models of cancer

The selection of efficacious anti-tumor monoclonal antibodies (MAbs) for biological applications is a lengthy and labor-intensive process. In vitro characterization of one hybridoma fusion may reveal large numbers of tumor antigen-specific hybridomas. Very often, many of these tumor-specific antibodies need to be assessed in vivo using several different murine xenograft tumor prevention models to determine biological efficacy. The production and purification of sufficient quantities of many antigen-specific hybridomas is time-consuming, and several months can pass between initial determination of MAb specificity and bioactivity. Moreover, many tumor-specific MAbs selected using in vitro binding studies have no in vivo anti-tumor efficacy. These studies describe an in vivo screening method either to eliminate non-efficacious MAbs or to rank-order several tumor-specific MAbs in an expeditious manner. Proof-of-concept studies were conducted using two hybridomas secreting fully characterized neutralizing human anti-tumor MAbs (CNTO MAbs). Nu-/nu- mice were injected with CNTO MAb-secreting hybridoma cells in Matrigel cell matrix, followed by injection of target human tumor cells 4 days later (when circulating CNTO MAbs were detected in serum). Both the tumor take-rate and the mean tumor volumes were reduced significantly in mice treated with CNTO MAbsecreting hybridomas compared with mice treated with non-antibody-secreting cells. A panel of human antitumor antigen-specific MAbs with unknown biological efficacy was then evaluated by this method. The hybridomas exhibited a varied pattern of anti-tumor protection, indicating that some hybrids were secreting neutralizing anti-tumor MAbs, while others appeared to be less efficacious. These studies demonstrate a rapid, biologically relevant "yes/no" in vivo screening method for the evaluation of anti-tumor antigen MAbs.     

Oncofetal antigen in Xiphophorus detected by monoclonal antibodies directed against melanoma-associated antigens

Monoclonal antibodies (MAbs) directed against Xiphophorus melanoma cells were developed and tested by indirect immunofluorescence and immunoperoxidase staining for reactivity with a panel of 15 allogeneic tissues and 12 allogeneic cell lines. The reactivity of such MAbs was restricted to melanoma cells from tumor biopsies and melanoma-derived cell lines. In addition, all embryonic cells of all histiotypes from developmental stages later than mid-organogenesis and from corresponding short term in vitro cultures reacted with these MAbs. In contrast, normal tissues and organs from adult fish displayed no reactivity, thus implying that the melanoma-associated antigens detected by the MAbs described are oncofetal antigens.     

Enhancement of colorectal tumor targeting using a novel biparatopic monoclonal antibody against carcinoembryonic antigen in experimental radioimmunoguided surgery.

Biparatopic CEA, carcinoembryonic antigen (MAb) was newly designed and tested as to whether it enhanced the accuracy of tumor detection by reducing non-specific binding in experimental radioimmunoguided surgery. Biparatopic MAb was prepared by using cross-linking of reduced Fab' fragments from PR1A3 and T84.66. Fifty-nine tumors from 2 human colorectal carcinoma cell lines with high (KM-12c) and low (Clone A) carcinoembryonic antigen (CEA) expression were successfully implanted subcutaneously on the backs of 42 nude mice. Tumors were localized using 125I-labeled MAbs: IgG, F(ab')(2) and Fab' of PR1A3, and biparatopic MAb of PR1A3 and T84.66. Radioactivity counted on a portable radioisotope detector correlated well with that counted on a gamma counter (p < 0.001). Accumulations of radioactivity in control mice without tumorigenesis were the greatest in PR1A3 IgG-pretreated mice and the least in biparatopic MAb-pretreated mice. Tumors of 2 cell lines did not differ in the distribution of radiolabeled MAbs. Localization indices of the tumor in various organs revealed 1.3 to 4.1 in PR1A3 IgG-pretreated mice, 2.4 to 6.6 in fragment MAbs of PR1A3-pretreated mice and 2 to 4.6 in biparatopic MAb-pretreated mice. Silver grains and immune staining were predominantly distributed in tumor cells of all types of MAb-pretreated mice. Sensitivity and specificity of tumor localization by radioimmunoguided surgery (RIGS) were the highest in the biparatopic MAb-pretreated mice (90.9% and 94.5%, respectively) and the least in the PR1A3 IgG-pretreated mice (50% and 72%). The biparatopic MAb using 2 anti-CEA MAbs against different epitopes achieved a great affinity and avidity with accurate localization of colorectal carcinoma in experimental radioimmunoguided surgery.     

Loss of HLA-A,B,C and de novo expression of HLA-D in colorectal cancer

One hundred primary colorectal carcinomas and 19 metastases were studied for the expression of HLA-A,B,C and HLA-D antigens using monoclonal antibodies (MAbs) against framework determinants and a sensitive immunoperoxidase technique on frozen sections. With MAb W6/32, 65 tumors were intensely stained for HLA-A,B,C; 22 showed a reduced staining intensity, in 5 carcinomas a minor or major subset of the tumor cells lacked HLA-A,B,C, and in 8 cases all tumor cells were unreactive. The loss of HLA-A,B,C was inversely correlated with the degree of differentiation. No relation was found to type, stage and site of the tumor, or to age and sex of the patients. With MAb 2.06, 58 tumors were HLA-D negative, 20 exhibited small positive foci, 19 showed a patchy staining pattern, and 3 were homogeneously HLA-D positive. There was no apparent correlation between HLA-D expression and any of the clinico-pathological features mentioned, nor was it related to the loss of HLA-A,B,C. About half of the metastases displayed the same staining pattern for HLA-A,B,C and -D as the respective primary tumors. The remainder differed from the primary carcinoma by gain or loss of HLA antigens.     

Generation and characterization of monoclonal antibodies to prostate secretory protein

Monoclonal antibodies (MAbs) were produced against a highly purified preparation of prostate secretory protein (PSP) isolated from normal seminal plasma. Fifteen antibodies were selected for further evaluation based on their strong reactivity and specificity for PSP. All the MAbs had a specificity for prostate epithelial cells and none reacted to any of a variety of normal tissues as determined by immunoperoxidase staining. Six of the MAbs were selected for further immunohistochemical evaluation based on their ability to recognize different antigenic determinants. Using competitive binding immunoassays, a variety of overlapping specificities were observed with at least 2 distinct epitopes identified. Although some staining variability was noted, the 6 antibodies, in general, gave the same pattern of tissue reactivity. Both the normal prostate and the benign prostate hyperplastic ductal epithelial cells stained intensely, with 78 to 100% and 50-100% of the cells staining, respectively. The number and often the staining intensity of the tumor cells decreased as the tumor became more undifferentiated. Approximately 40 to 100% and 15 to 70% of the tumor cells stained in the moderately-differentiated and well-differentiated carcinoma tissues, respectively, whereas either no staining was observed or less than 20% of the tumor cells stained in the poorly-differentiated and undifferentiated tumors. Most of the metastatic prostate tumors showed either no staining or scattered staining in a few cells (i.e., less than 20%).     

The EORTC Melanoma Group exchange program: evaluation of a multicenter monoclonal antibody study

In the framework of the European Organisation for Research and Treatment of Cancer (EORTC), the Immunology and Pathology Subgroups of the Malignant Melanoma Cooperative Group undertook a large multicenter monoclonal antibody (MAb) study. Fourteen laboratories from 7 European countries tested a panel of 23 MAbs for immunohistological staining reactivity for malignant and non-malignant lesions involving the melanocytic lineage. A standardized immunoperoxidase procedure was used and the results were evaluated using a standard protocol and data evaluation form developed in collaboration with the EORTC Data Center. According to this analysis, the antibodies in the panel could be classified into 3 main groups. The first group of MAbs includes those antibodies which stained the majority (greater than 80%) of all primary tumors, irrespective of their Breslow thickness and the majority of metastatic lesions. In addition, these MAbs stained a high percentage of cells within a given lesion. Several antibodies of Group I were likewise reactive with the majority of naevoblasts and with normal melanocytes. The second group of MAbs included antibodies reacting only with a limited number of primary melanomas and metastatic lesions. Antibodies of Group II reacted only weakly, if at all, with normal melanocytes or naevocytes. The percentage of cells within a malignant lesion stained by these MAbs was always rather low. The MAb group III detected surface structures whose expression appeared to be related to tumor progression; they did not react or reacted only weakly with naevi, and they all reacted with a small number of early primary melanomas (less than 0.75 mm). The number of lesions stained increased with increasing Breslow thickness. Our study suggests that the application of a panel of well defined MAbs might be of diagnostic and prognostic value in evaluating malignant melanoma.     

Defective vasculature in fibronectin-receptor-deficient cho cell tumors in nude mice

The level of expression of the α5/β1 integrin fibronectin receptor(FnR) strongly affects the growth rate of CHO cell tumors in nude mice. Here we report that α5/β1 expression also influences the organization of the tumor vasculature. Tumors formed from CHO clones defective in FnR expression have a leaky vasculature that gives them a hemorrhagic appearance. Tumors from wild-type CHO cells, or from FnR-deficient CHO clones transfected with constructs coding for the α5 integrin subunit, have an intact, non-leaky vasculature. In tumors from FnR-deficient cells, the endothelial lining of blood vessels is sparse, and red cells and plasma proteins can be detected in the tumor parenchyma. In tumors from cells expressing the α5/β1 FnR, tumor vessels are circumscribed by a lining of von-Willebrand-factor-positive endothelial cells, and blood cells and proteins are confined to the vessel lumina. Thus, the level of expression of the α5/β1 FnR in the tumor parenchymal cells can influence the development of tumor vasculature. Since α5/β1 is vital to the organization of the extracellular matrix, one possibility is that altered matrix assembly contributes to the defective vascularization seen in α5/β1 -deficient tumors.     

Monoclonal-antibodies 5g8 and 2h6 are complementary immunohistochemical markers of lung carcinomas

Two murine monoclonal antibodies (MAbs) 5G8 and 2H6 were generated by fusing spleen cells obtained from mice immunized with human fetal tissue extract (14 weeks, Nonidet P-40 membrane fraction) from the early first trimester, followed by a booster injection of a lung cancer cell line. The MAb 5G8 recognized antigens with a molecular weight of 90, 50, 20 kDa, was neuraminidase-resistant and showed cross-reactivity with both carcinoembryonic antigen and non-specific cross-reacting antigen. The MAb 2H6 reacted with a sialomucin whose molecular weight was more than 1000 kDa and was different from previously known tumor associated-marker antigens. The distribution of the MAb-recognizing antigens in various tissues was investigated immunohistochemically. In malignant epithelial tumors of the lung, acinar adenocarcinoma and bronchioloalveolar carcinoma were positive for both MAbs; papillary adenocarcinoma, squamous cell carcinoma, and adenoid cystic carcinoma were positive only for MAb 5G8; solid carcinoma with mucin formation, small cell carcinoma, and large cell carcinoma were positive only for MAb 2H6. The combination of the two MAbs covered almost all histological types of lung carcinomas (23 of 24 cases) except carcinoid tumors. MAbs 5G8 and 2H6 reacted also with a restricted number of adenocarcinomas of the other organs. MAb 5G8 did not react with normal fetal or adult tissues, except for metaplastic gastric mucosa, acinar cells of the breast and leucocytes, whereas MAb 2H6 reacted with the surface epithelium of the stomach and colon, the Brunner gland of the duodenum and uterine cervix as well as the epithelium of the fetal digestive tract. Thus, MAb 2H6 which recognized oncofetal sialomucin, played a complementary role to MAb 5G8 as a CEA-related MAb in the immunohistochemical diagnosis of lung carcinomas.     

Re-targeting of human lymphocytes expressing the T-cell receptor gamma/delta to ovarian carcinoma cells by the use of bispecific monoclonal antibodies

TcR gamma/delta+ lymphocytes represent a small subset homogeneously composed of cytolytic T cells displaying unique motility and homing properties. Since the lytic machinery of these cells can be efficiently triggered by monoclonal antibodies (MAbs) directed to the TcR gamma/delta, such MAbs were used for the construction of bispecific MAbs in conjunction with an MAb specific for ovarian carcinoma cells. Hybrid hybridomas were obtained by fusing the Mov19 MAb (IgG2a)-producing hybridoma with either GI (IgG2a) or A13 (IgG1) hybridomas, secreting MAbs specific for 2 peripheral blood subsets of TcR gamma/delta+ lymphocytes. Hybrid hybridomas producing bispecific MAbs were screened according to their ability to induce ovarian carcinoma (IGROVI) target cell lysis by GI+ or A13+ T cell clones, respectively. The GI-derived GM33.9 bispecific MAb induced selective lysis of Mov19+ ovarian carcinoma target cells only by GI+ clones, whereas the A13-derived AM18.4 MAb was effective only in combination with A13+ clones. Neither the anti-TcR gamma/delta nor the Mov19 parental MAbs (used alone or in combination) induced target-cell lysis. The hybrid nature (IgG1/IgG2a) of the AM18.4 bispecific MAb was indicated by 2-color immunofluorescence experiments. Thus, both ovarian carcinoma and A13+ effector cells were double stained by AM18.4 bispecific MAb followed by PE-conjugated anti-IgG1 and FITC-conjugated anti-IgG2a second reagents. Polyclonal TcR gamma/delta+ cells were obtained by direct stimulation of peripheral blood mononuclear cells with Sepharose bead-conjugated anti-TcR gamma/delta MAbs and IL-2. The lines so obtained contained more than 90% of TcR gamma/delta+ cells after 4 weeks of culture, with an increase in TcR gamma/delta+ cell numbers ranging from 200 to 1,000-fold. These TcR gamma/delta+ cell lines efficiently lysed ovarian carcinoma target cells in the presence of bispecific MAb and may therefore represent a suitable source of effector cells for induction of ovarian carcinoma cell lysis.