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We describe a novel fluorescent method for the detection of receptors for chimeric proteins in tissue sections. The technique was developed using a recombinant human insulin-like growth factor (IGF-1) chimera, bearing six additional histidine residues at the carboxy-terminal end (IGF-1-His). We demonstrated that dehydration of the tissue sections was detrimental for binding and that its prevention dramatically increased sensitivity. The specificity of IGF-1-His interaction was shown by gradual abolition of the fluorescent signal in the presence of increasing concentrations of IGF-1. Combining immunofluorescence with in situ ligand binding, we showed that IGF-1-His binding corresponded to the IGF-1 receptor (IGFR-1) distribution in human fetal kidney. Moreover, incubation of the tissue sections with an anti-IGFR-1 blocking antibody abolished IGF-1-His binding, demonstrating that the interaction was mediated by the IGFR-1. The method was also used to localize the IGFR-1 in E18 rat embryo sagittal sections. The IGF-1-His binding pattern was observed in brain, cartilage, lung, skin, heart, diaphragm, and tongue, and paralleled the previously reported IGFR-1 distribution. We believe that this new non-isotopic in situ ligand binding method will facilitate rapid and accurate localization of receptors in tissue sections.

HER2 is a ligand-less member of the human epidermal growth factor receptor or ErbB family of tyrosine kinases. In normal biological systems, HER2 functions as a co-receptor for a multitude of epidermal growth factor-like ligands that bind and activate other HER family members. HER2 overexpression is observed in a number of human adenocarcinomas and results in constitutive HER2 activation. Specific targeting of these tumors can be accomplished with antibodies directed against the extracellular domain of the HER2 protein. One of these antibodies, 4D5, has been fully humanized and is termed trastuzumab (Herceptin; Genentech, San Francisco, CA). Treatment of HER2-overexpressing breast cancer cell lines with trastuzumab results in induction of p27KIP1 and the Rb-related protein, p130, which in turn significantly reduces the number of cells undergoing S-phase. A number of other phenotypic changes are observed in vitro as a consequence of trastuzumab binding to HER2-overexpressing cells. These phenotypic changes include downmodulation of the HER2 receptor, inhibition of tumor cell growth, reversed cytokine resistance, restored E-cadherin expression levels, and reduced vascular endothelial growth factor production. Interaction of trastuzumab with the human immune system via its human immunoglobulin G1 Fc domain may potentiate its antitumor activities. In vitro studies demonstrate that trastuzumab is very effective in mediating antibody-dependent cell-mediated cytotoxicity against HER2-overexpressing tumor targets. Trastuzumab treatment of mouse xenograft models results in marked suppression of tumor growth. When given in combination with standard cytotoxic chemotherapeutic agents, trastuzumab treatment generally results in statistically superior antitumor efficacy compared with either agent given alone. Taken together, these studies suggest that the mechanism of action of trastuzumab includes antagonizing the constitutive growth-signaling properties of the HER2 system, enlisting immune cells to attack and kill the tumor target, and augmenting chemotherapy-induced cytotoxicity.

The HER2/neu oncogene product, p185(HER2/neu), is overexpressed on the surface of many human breast cancers. Strains of transgenic mice have been developed that express the rat neu oncogene in mammary epithelial cells and develop spontaneous mammary tumors that overexpress p185neu. This model provides an ideal system for testing interventions to prevent tumor development. In this study, we immunized neu-transgenic mice with a vaccine consisting of the extracellular domain of p185neu (NeuECD). Immunized mice developed Neu-specific humoral immune responses, as measured by circulating anti-Neu antibodies in their sera, and cellular immune responses, as measured by lymphocyte proliferation to NeuECD in vitro. In addition, the subsequent development of mammary tumors was significantly lower in immunized mice than in controls and vaccine treatment was associated with a significant increase in median survival.

Thrombopoietin (TPO) is a hematopoietin important for megakaryocyte proliferation and production of blood platelets. We sought to characterize how TPO binds and activates its receptor, myeloproliferative leukemia virus receptor. The erythropoietin-like domain of TPO (TPO1-153) has been fused to the gIII coat protein of M13 bacteriophage. Forty residues were chosen for mutation to alanine using the criteria that they were charged residues or predicted to be solvent-exposed, based on a homology model. Phage enzyme-linked immunosorbent assay was used to determine affinities for binding to both the TPO receptor and five anti-TPO1-153 monoclonal antibodies. Mutations at mostly positively charged residues (Asp8, Lys14, Lys52, Lys59, Lys136, Lys138, Arg140) caused the greatest reduction in receptor-binding affinity. Most of these residues mapped to helices-1 and -4 and a loop region between helix-1 and helix-2. Two of the monoclonal antibodies that blocked TPO binding and bioactivity had determinants in helix-4. In contrast, the other three monoclonal antibodies, which were effective at blocking TPO activity but did not block initial binding of TPO to its receptor, had epitopes predominantly on helix or 3. These results suggest that TPO has two distinct receptor-binding sites that function to dimerize TPO receptors in a sequential fashion.

Alterations in the expression of the epidermal growth factor (EGF) receptor ErbB family are frequently encountered in a number of human cancers. Two of these receptors, ErbB3 and ErbB4, are known to bind a family of related proteins termed heregulins (HRGs) or neu differentiation factors. In biologically relevant systems, interaction of HRG with ErbB3 or ErbB4 results in the transactivation of ErbB2. In this report, we show that ErbB2 is a critical component in mediating HRG responsiveness in a panel of human breast and ovarian tumor cell lines. Because HRGs have been reported to elicit diverse biological effects on cultured cells, including growth stimulation, growth inhibition, and induction of differentiation, we systematically examined the effect of rHRG beta 1 on tumor cell proliferation. HRG binding studies were performed with a panel of breast and ovarian tumor cell lines expressing a range of levels of ErbB2. The biological responses to HRG were also compared to EGF and to the growth-inhibitory anti-ErbB2 antibody, 4D5. In most cases, HRG stimulation of DNA synthesis correlated with positive effects on cell cycle progression and cell number and with enhancement of colony formation in soft agar. On each cell line tested, the HRG effects were distinguishable from EGF and 4D5. Our findings indicate that HRG induces cell proliferation in a number of tumor cell lines. In addition, we show that methods for measuring cell proliferation, as well as experimental conditions, are critical for determining HRGs effect on tumor cell growth in vitro.

Transforming growth factor beta (TGF-beta) is a multifunctional peptide that controls proliferation, differentiation, and other functions in a variety of cell types. Transforming growth factor beta activities have been implicated in a variety of diseased states including arthritis, prostate cancer, and AIDS, and in the repair of tissue injury caused by trauma, burns, and surgery. We describe the development and characterization of novel murine monoclonal antibodies (MAbs) to the latency-associated peptide (LAP) of TGF-beta 1, and the subsequent development of an ELISA for the detection and quantitation of TGF-beta 1-LAP in buffer and serum matrices. Fusion of immune splenocytes with myeloma cells yielded 576 hybridomas, 110 of which were antibody secreting. Five were selected for extensive characterization. Clinically, the MAbs described here should be valuable for studying potentially abnormal production and/or function of the LAP, and its relationship to TGF-beta.

The most potent known agonist for the natriuretic peptide receptor-B (NPR-B)/guanylyl cyclase-B is C-type natriuretic peptide (CNP). A homologous ligand-receptor system consists of atrial natriuretic peptide (ANP) and NPR-A/guanylyl cyclase-A. A third member of this family is NPR-C, a non-guanylyl cyclase receptor. Monoclonal antibodies were raised against NPR-B by immunizing mice with a purified receptor-IgG fusion protein consisting of the extracellular domain of NPR-B and the Fc portion of human IgG-gamma 1. One monoclonal antibody, 3G12, did not recognize NPR-A or NPR-C and bound to human and rat NPR-B. CNP binding to NPR-B and stimulation of cGMP synthesis were inhibited by 3G12. With cells isolated from either the media or adventitia layers of rat thoracic aorta, 3G12 did not interfere with ANP-stimulated cGMP synthesis, but it inhibited CNP-stimulated cGMP levels in cells from both layers. CNP (IC50 = 10 nM) and ANP (IC50 = 1 nM) caused relaxation of phenylephrine-contracted rat aortic rings. 3G12 caused a marked increase in the IC50 for CNP, from 10 nM to 140 nM, but failed to affect ANP-mediated relaxation. Therefore, our results for the first time demonstrate that CNP relaxes vascular smooth muscle by virtue of its binding to NPR-B.

The cytotoxic effects of TNF on malignant cells are known to be mediated through high affinity surface receptors. The precise mechanism by which transformed cells are selectively killed by the activation of these receptors is yet unknown, but several intracellular signaling pathways are known to be involved. Phospholipase A2 activation by TNF-alpha has been shown to be important in the transduction of signals leading to cell death. We have used monitoring of extracellular acidification rate as a measure of cellular metabolism to follow the early time course of TNF effects on a human leukemic T cell line (CEM-SS cells). CEM-SS cells were relatively resistant to TNF cell killing but TNF caused an early stimulation of metabolism within 2-4 hr, followed by a suppression of metabolic activity occurring over 20 hr. In contrast, a TNF sensitive subclone of CEM cells (C1Ca) showed a rapid and dramatic decrease in metabolic activity corresponding to cytotoxicity within 18 hr. It was discovered that cupric o-phenanthroline markedly potentiated the effects of TNF on the resistant CEM-SS cells leading to cell death. This observation was specific for copper because ferric o-phenanthroline was without effect at the same concentration. The copper cytotoxic effect was shown to be mediated through the TNF-R1 receptor and independent of phospholipase A2 signaling.

Approximately 30% of human breast and ovarian cancers have amplification and/or overexpression of HER-2/neu gene which encodes a cell surface growth-factor receptor. Overexpression of this receptor, p185HER-2/neu, is associated with poor outcome and may predict clinical response to chemotherapy. Antibodies to HER-2/neu receptor have a cytostatic effect in suppressing growth of cells with overexpression of p185HER-2/neu. To elicit a cytocidal effect, therapy with antireceptor antibody was used in combination with the DNA-damaging drug, cisplatin, and this combined treatment produced a synergistic decrease in cell growth. In addition, antibody mediated an increased sensitivity to cisplatin in drug-resistant ovarian carcinoma cells containing multiple copies of HER-2/neu gene. To evaluate the mechanism for this synergy, unscheduled DNA synthesis was measured in cancer cells using incorporation of [3H]thymidine and autoradiography, and formation and repair of cisplatin-induced DNA adducts was also measured. Treatment with cisplatin led to a marked, dose-dependent increase in unscheduled DNA synthesis which was significantly reduced by combined treatment with antireceptor antibody in HER-2/neu-overexpressing cells. Therapy with antibody to HER-2/neu receptor also led to a 35-40% reduction in repair of cisplatin-DNA adducts after cisplatin exposure and, as a result, promoted drug-induced killing in target cells. This phenomenon which we term receptor-enhanced chemosensitivity may provide a rationale for more selective targeting and exploitation of overexpressed growth factor receptors in cancer cells, thus leading to new strategies for clinical intervention.

The heregulin/neu differentiation factor gene products were purified and cloned based on their ability to stimulate the phosphorylation of a 185-kDa protein in human breast carcinoma cell lines known to express erbB2. However, not all cells that express erbB2 respond to heregulin, indicating that other components besides erbB2 may be required for heregulin binding. Cells that are transfected with the closely related receptor, erbB3, display a single class of lower affinity heregulin binding sites than has been previously observed on breast carcinoma cell lines. Little or no stimulation of tyrosine phosphorylation in response to heregulin occurs in cells that are transfected with erbB3 alone. Transfection of cells with erbB3 and erbB2 reconstitutes a higher affinity binding receptor, which is also capable of generating a tyrosine phosphorylation signal in response to heregulin. A monoclonal antibody to erbB2 will inhibit heregulin activation of tyrosine phosphorylation and binding in cells transfected with both receptors but not with erbB3 alone. In cells expressing erbB2 and erbB3, both proteins become tyrosine-phosphorylated upon interaction with heregulin. Direct interaction between heregulin and the two proteins was demonstrated by chemical cross-linking experiments using 125I-heregulin followed by immunoprecipitation with antibodies specific for erbB2 or erbB3.

TGF-beta effects on angiogenesis, stroma formation, and immune function suggest its possible involvement in tumor progression. This hypothesis was tested using the 2G7 IgG2b, which neutralizes TGF-beta 1, -beta 2, and -beta 3, and the MDA-231 human breast cancer cell line. Inoculation of these cells in athymic mice decreases mouse spleen natural killer (NK) cell activity. Intraperitoneal injections of 2G7 starting 1 d after intraperitoneal inoculation of tumor cells suppressed intraabdominal tumor and lung metastases, whereas the nonneutralizing anti-TGF-beta 12H5 IgG2a had no effect. 2G7 transiently inhibited growth of established MDA-231 subcutaneous tumors. Histologically, both 2G7-treated and control tumors were identical. Intraperitoneal administration of 2G7 resulted in a marked increase in mouse spleen NK cell activity. 2G7 did not inhibit MDA-231 primary tumor or metastases formation, nor did it stimulate NK cell-mediated cytotoxicity in beige NK-deficient nude mice. Finally, serum-free conditioned medium from MDA-231 cells inhibited the NK cell activity of human blood lymphocytes. This inhibition was blocked by the neutralizing anti-TGF-beta 2G7 antibody but not by a nonspecific IgG2. These data support a possible role for tumor cell TGF-beta in the progression of mammary carcinomas by suppressing host immune surveillance.

TNF-alpha can enhance the proliferation of human thymocytes stimulated by the comitogen Con A. To determine which of the two different TNF receptors is responsible for signaling this cellular response, we investigated the proliferation of human thymocytes in response to agonistic antibodies specific for the two TNF receptor types. In contrast to previously examined TNF activities in human cells, thymocyte proliferation was stimulated in response to rabbit polyclonal antibodies directed against the 75-kDa TNF receptor (TNF-R2), but not those directed against the 55-kDa TNF receptor (TNF-R1). Lymphotoxin (TNF-beta) was also shown to stimulate human thymocyte proliferation, demonstrating that TNF-beta can initiate a biologic response that is mediated by TNF-R2. TNF-R2-mediated T-cell proliferation was not restricted to the immature T cells within the thymus, as the anti-TNF-R2 antibodies also stimulated the proliferation of peripheral T cells. As a first step toward identifying a specific agonist of TNF-R2 with therapeutic potential, 10 anti-TNF-R2 mAb were examined for potential agonist activity. Nine of these significantly stimulated human thymocyte proliferation with maximal responses ranging from twofold to significantly greater than that obtained with TNF-alpha by itself.

The binding properties of seven CD4-blocking monoclonal antibodies raised against recombinant gp120 of human immunodeficiency virus type 1 strain MN (HIV-1MN) and two CD4-blocking monoclonal antibodies to recombinant envelope glycoproteins gp120 and gp160 of substrain IIIB of HIVLAI were analyzed. With a panel of recombinant gp120s from seven diverse HIV-1 isolates, eight of the nine antibodies were found to be strain specific and one was broadly cross-reactive. Epitope mapping revealed that all nine antibodies bound to epitopes located in the fourth conserved domain (C4) of gp120. Within this region, three distinct epitopes could be identified: two were polymorphic between HIV-1 strains, and one was highly conserved. Studies with synthetic peptides demonstrated that the conserved epitope, recognized by antibody 13H8, was located between residues 431 and 439. Site-directed mutagenesis of gp120 demonstrated that residue 429 and/or 432 was critical for the binding of the seven antibodies to gp120 from HIV-1MN. Similarly, residues 423 and 429 were essential for the binding of monoclonal antibody 5C2 raised against gp120 from HIV-1IIIB. The amino acids located at positions 423 and 429 were found to vary between strains of HIV-1 as well as between molecular clones derived from the MN and LAI isolates of HIV-1. Polymorphism at these positions prevented the binding of virus-neutralizing monoclonal antibodies and raised the possibility that HIV-1 neutralization serotypes may be defined on the basis of C4 domain sequences. Analysis of the binding characteristics of the CD4-blocking antibodies demonstrated that their virus-neutralizing activity was directly proportional to their gp120-binding affinity. These studies account for the strain specificity of antibodies to the C4 domain of gp120 and demonstrate for the first time that antibodies to this region can be as effective as those directed to the principal neutralizing determinant (V3 domain) in neutralizing HIV-1 infectivity.

IgE antibodies bind to specific high-affinity receptors on mast cells, leading to mast cell degranulation and release of mediators, such as histamine, which produce symptoms associated with allergy. Hence, anti-IgE antibodies that block binding of IgE to its high-affinity receptor are of potential therapeutic value in the treatment of allergy. These antibodies must also not bind to IgE once it is bound to the receptor because this would trigger histamine release. This study describes the humanization of a murine antibody, MaE11, with these characteristics. Variants of the humanized antibody were evaluated to probe the importance of framework residues on antibody binding and to determine which charged residues in the CDR interacted with IgE. We found that only five changes in human framework residues were required to provide for binding comparable to that of the original murine antibody.

A polyclonal chicken antiserum against purified 32-kilodalton (kDa) recombinant inhibin-A (rh-InhA) and two monoclonal antibodies (mAb) against either rh-InhA (11B5) or 28-kDa recombinant activin-A (rh-ActA; 9A9) were used to develop three sensitive InhA enzyme-linked immunosorbent assays (ELISAs). The sensitivity of an ELISA using affinity-purified chicken anti-rh-InhA (Ck) for both coat and capture (Ck/Ck) averaged 78 +/- 3 pg/ml, while the mAb/Ck ELISAs (11B5/Ck or 9A9/Ck) averaged 100 +/- 6 pg/ml in a 10% serum matrix, with intra-and interassay coefficients of variation of 2-5% and 8-10%, respectively, for all assays. The ELISA formats did not cross-react with purified rh-ActA or recombinant human transforming growth factor-beta 1 or detect any immunoreactive proteins in medium conditioned by cell lines expressing rh-ActA or recombinant human transforming growth factor-beta 1. The Ck/Ck ELISA detected significant amounts of immunoreactivity in medium from cells expressing the free alpha-subunit of inhibin and recombinant inhibin-B (rh-InhB). In contrast, the mAb/Ck ELISAs showed no cross-reactivity to medium conditioned by these two cell lines. All three ELISA formats detected rh-InhA added to either human or rat serum in vitro or serum from rats injected with rhInhA. The Ck/Ck and 9A9/Ck ELISAs successfully quantitated inhibin in sera from patients undergoing ovulation induction and in rats (with or without sc administration of pregnant female serum gonadotropin). The 11B5/Ck ELISA appeared to be specific for the 32-kDa form of inhibin, while the 9A9/Ck ELISA was useful in quantitating inhibin-A in biological fluids, with little cross-reactivity to free alpha-chain or inhibin-B.

Cloned sequences encoding a truncated form of the HER2 receptor were obtained from cDNA libraries derived from two HER2-overexpressing human breast cancer cell lines, BT-474 and SK-BR-3. The 5' 2.1 kb of the encoded transcript is identical to that of full-length 4.6-kb HER2 transcript and would be expected to produce a secreted form of HER2 receptor containing only the extracellular ligand binding domain (ECD). The 3' end of the truncated transcript diverges 61 nucleotides before the receptor's transmembrane region, reads through a consensus splice donor site containing an in-frame stop codon, and contains a poly(A) addition site, suggesting that the truncated transcript arises by alternative RNA processing. S1 nuclease protection assays show a 40-fold variation in the abundance of the truncated 2.3-kb transcript relative to full-length 4.6-kb transcript in a panel of eight HER2-expressing tumor cell lines of gastric, ovarian, and breast cancer origin. Expression of this truncated transcript in COS-1 cells produces both secreted and intracellular forms of HER2 ECD; however, immunofluorescent labeling of HER2 ECD protein in MKN7 tumor cells that natively overexpress the 2.3-kb transcript suggests that transcriptionally generated HER2 ECD is concentrated within the perinuclear cytoplasm. Metabolic labeling and endoglycosidase studies suggest that this HER2 ECD (100 kDa) undergoes differential trafficking between the endoplasmic reticulum and Golgi compartments compared with full-length (185-kDa) HER2 receptor. Transfection studies indicate that excess production of HER2 ECD in human tumor cells overexpressing full-length HER2 receptor can result in resistance to the growth-inhibiting effects of anti-HER2 monoclonal antibodies such as muMAb4D5. These findings demonstrate alternative processing of the HER2 transcript and implicate a potentially important growth regulatory role for intracellularly sequestered HER2 ECD in HER2-amplified human tumors.

Ten monoclonal antibodies prepared against a soluble, recombinant form of gp160, derived from the IIIB isolate of HIV-1, were characterized. Four of the antibodies neutralized HIV-1IIIB infectivity in vitro, three blocked the binding of recombinant gp120 to CD4, three were reactive with gp41, and one preferentially reacted with an epitope on gp120 within the gp160 precursor. All three CD4 blocking antibodies bound to distinct epitopes, with one mapping to the C1 domain, one mapping to the C4 domain, and one reactive with a conformation-dependent, discontinuous epitope. Of these, the antibody reactive with the discontinuous epitope exhibited neutralizing activity against homologous and heterologous strains of HIV-1. The binding of these monoclonal antibodies to a panel of seven recombinant gp120s prepared from diverse isolates of HIV-1 was measured, and monoclonal antibodies with broad cross reactivity were identified. The epitopes recognized by 7 of the 10 monoclonal antibodies studied were localized by their reactivity with synthetic peptides and with fragments of gp120 expressed as fusion proteins in a lambda gt-11 gp160 epitope library.

Most of the physiological actions of atrial natriuretic peptide (ANP) may be attributed to activation of the natriuretic peptide receptor-A (NPR-A) guanylyl cyclase. We report here that truncation of the NPR-A cytoplasmic domain results in increased expression of cell surface ANP binding sites. The truncated receptor exhibited a hyperbolic time course for ANP binding and had a high affinity for [125I]hANP, Kd = 8 pM. Cells expressing truncated NPR-A were used as an immunogen to obtain monoclonal antibodies against the native conformation of the extracellular domain. These antibodies were used to select for high levels of stable NPR-A expression in 293 cells, by fluorescence-activated cell sorting. Disuccinimidyl suberate cross-linked [125I]ANP to 135-kDa NPR-A on intact cells. Monoclonal antibody immunoprecipitation of 35S-labeled proteins revealed NPR-A size heterogeneity, with 135- and 125-kDa species. A synthetic peptide antibody directed against the extracellular domain immunoprecipitated 125-kDa NPR-A, but recognized both sizes of receptor by Western blotting. The 125-kDa NPR-A did not bind to or cross-link ANP. NPR-A size variants were expressed on the cell surface, and heterogeneity was removed by deglycosylation with protein:N-glycosidase F. Our results suggest that the degree of N-linked glycosylation of the NPR-A extracellular domain influences the ability to bind ANP.

An expression plasmid encoding the human 75-kDa tumor necrosis factor (TNF) type 2 receptor (TNF-R2) was constructed and used to generate a stable human cell line (293/TNF-R2) overexpressing TNF-R2. Ligand binding analysis revealed high affinity binding (Kd = 0.2 nM) with approximately 94,000 +/- 7,500 sites/cell for 125I-TNF-alpha and approximately 5-fold lower affinity for TNF-beta (Kd = 1.1 nM) with 264,000 +/- 2,000 sites/cell. Cross-linking of 125I-TNF-alpha and 125I-TNF-beta to 293/TNF-R2 cells yielded predominant complexes with apparent molecular weights of 211,000 for TNF-alpha and 205,000 and 244,000 for TNF-beta, suggesting these complexes contain two or three TNF-R2 molecules. Immunoprecipitation of TNF-R2 from 32P-labeled 293/TNF-R2 cells demonstrated that the receptor is phosphorylated. The majority (97%) of 32Pi incorporation was found in serine residues with a very low level of incorporation (3%) in threonine residues. TNF-alpha treatment of 293/TNF-R2 cells did not significantly affect the degree or pattern of phosphorylation. Cell surface-bound 125I-TNF-alpha was slowly internalized by the 293/TNF-R2 cell line with a t1/2 = 25 min. Shedding of the extracellular domain of TNF-R2 was induced by 4 beta-phorbol 12-myristate 13-acetate but not by TNF-alpha or TNF-beta.

A comprehensive mutational analysis was used to analyze the side-chains on human growth hormone (hGH) important for binding 21 different anti-hGH mouse monoclonal antibodies (MAbs) whose equivalent concentrations for 50% binding (EC50) ranged from approximately 10(7) to 3 x 10(10) M-1. A combination of homolog- and alanine-scanning mutagenesis coupled with a robot-aided enzyme-linked immunosorbent assay were used to create high resolution "functional epitopes" for each MAb. Every functional epitope mapped to at least two polypeptide segments of hGH that were close together in the folded protein to form a patch. Although these patches sometimes overlapped, each was different indicating no two MAbs bound identically to hGH. The MAbs bound to determinants in loops and helices that were generally most accessible to a 9 A radius probe. Only a few side-chains dominated each functional epitope and these tended to be Arg greater than Pro greater than Glu approximately Asp approximately Phe approximately Ile (Ala, Cys and Trp were not tested). Our studies indicate that most of the accessible surface of hGH is potentially antigenic in the mouse and suggest that functional epitopes are dominated by fewer side-chains than may be in the contact epitope.

Paraffin wax sections of 70 surgically resected colorectal adenocarcinomas were examined for the overexpression of HER2/c-erbB-2 oncoprotein using three different specific antibodies and the avidin-biotin immunoperoxidase technique. The patients included 38 men and 32 women aged between 47 and 80 years. The tumours were derived from various parts of the large intestinal tract, and represented all three stages of Dukes' classification and the three histological grades of differentiation. Many tumour sections also included adjacent normal or transitional mucosa. Eight tubular adenomas found in the colectomy specimens in association with some carcinomas were also examined. No positive membrane staining was seen in any of the 70 carcinomas, four adenomas, two hyperplastic polyps, nor in the adjacent normal or transitional mucosa. It is suggested that the overexpression of c-erbB-2 gene product is unlikely to be as common and as pronounced in colorectal adenocarcinoma as it is in ductal carcinoma of the breast.

The extracellular portion of the type 1 (p55) and type 2 (p75) tumor necrosis factor (TNF) receptors contains a repetitive amino acid sequence pattern of four cysteine-rich domains (CRDs). This pattern is found also in several other cell surface proteins, including the p75 nerve growth factor receptor and the CD40, 4-1BB, OX40, Fas, and CD27 antigens. To investigate whether CRDs play a role in TNF binding, we have constructed soluble variants of the extracellular portion of human type 1 TNF receptor (sTNFR1), in which CRD1 (N-terminal) or CRD4 (C-terminal) was deleted by mutagenesis. These variants or a wild type sTNFR1 were linked in their C terminus to the hinge and Fc portion of IgG1 heavy chain to create sTNFR1-IgG chimeras (immunoadhesins). Deletion of either CRD1 or -4 did not cause any major perturbations in the structure of the sTNFR1 variants, as evidenced by their efficient expression and secretion from transfected cells, and by their binding to conformation-dependent monoclonal antibodies that recognize diverse epitopes on sTNFR1. The wild type sTNFR1 immunoadhesin exhibited high affinity binding to TNF alpha (Kd = 65 pM) and TNF beta (Kd = 640 pM). Deletion of CRD4 resulted in about a 10-fold reduction in affinity for TNF alpha (Kd = 660 pM) and for TNF beta (Kd = 5.7 nM). In contrast, deletion of CRD1 resulted in a complete loss of binding to TNF alpha and to TNF beta. These results indicate that CRD4 is important but not necessary for TNF binding, while CRD1 is required. In addition, the results suggest some overlap between the TNFR1 binding sites for TNF alpha and TNF beta, despite low amino acid sequence homology between these cytokines.

Recently, we reported that ovariectomy (OVX)-induced FSH hypersecretion can be elicited in hypophysectomized rats bearing renal pituitary allografts isolated from direct hypothalamic intervention. The possible role of the FSH-stimulating protein, activin-B, in eliciting this response was investigated using passive immunoneutralization with a monoclonal antibody (MAb) generated against activin-B. Other hypophysectomized/pituitary-grafted (H/G) rats serving as controls received an equivalent amount of a MAb incapable of neutralizing the biological actions of activin-B. Administration of increasing doses of the MAb prior to OVX dose-dependently suppressed serum FSH levels 12 h after OVX. Less consistent effects were observed 24 h after OVX even though an additional injection of the MAb was given 12 h after OVX in one study. Since it has been postulated that the periovulatory increase in additional injection of the MAb was given 12 h after OVX in one study. Since it has been postulated that the periovulatory increase in FSH secretion on estrus which is important for recruitment of follicles is a hypothalamic-independent phenomena, a separate experiment was performed in order to ascertain whether a local regulatory mechanism involving activin-B is operative on estrus. As in the preceding study using H/G rats, administration of the activin-B MAb on the evening of proestrus significantly attenuated serum FSH rises early on estrus. These results are consonant with the evolving concept that an important mechanism exists within the anterior pituitary proper for regulation of FSH secretion that involves the autocrine actions of activin-B.

An expression plasmid encoding the extracellular portion of the human tumor necrosis factor (TNF) type 1 receptor (TNF-R1) was constructed and used to generate a stable cell line secreting soluble TNF-R1 (sTNF-R1). The sTNF-R1 was purified, and its biochemical properties and its interactions with human TNF-alpha were examined. SDS-PAGE resolved the purified sTNF-R1 into three bands of approximate Mr 24,200, 28,200, and 32,800. Sedimentation equilibrium analysis gave a molecular weight of 25,000 for sTNF-R1 whereas the molecular weight obtained by gel filtration chromatography was approximately 55,000-60,000. Scatchard analysis of [125I]TNF-alpha binding to sTNF-R1 revealed high-affinity binding (Kd = 93 pM), comparable to that observed for the intact receptor on whole cells. Competitive binding experiments showed that sTNF-R1 has a 50-60-fold higher affinity for TNF-alpha than for TNF-beta, in contrast to the equal affinities of TNF-alpha and TNF-beta for the full-length TNF-R1 transiently expressed in mammalian cells. The sTNF-R1 was found to block the cytotoxicity of TNF-alpha and TNF-beta on a murine L-M cell assay. The sizes of the sTNF-R1.TNF-alpha complex determined by gel filtration chromatography and sedimentation equilibrium were approximately 141 and 115 kDa, respectively. The stoichiometry of the complex was examined by Scatchard analysis, size-exclusion chromatography, HPLC separation, amino acid composition, sequence analysis, and sedimentation equilibrium. The data from these studies suggest that at least two molecules of sTNF-R1 can bind to a single TNF-alpha trimer.(ABSTRACT TRUNCATED AT 250 WORDS)

The HER2 protooncogene encodes a growth factor receptor-like transmembrane protein tyrosine kinase (p185HER2) whose ligand remains to be fully characterized. The overexpression of p185HER2 is implicated in aggressive forms of breast and ovarian cancers. The role of p185HER2 in aggressive malignancy, as well as its cell surface localization, makes it an attractive target for therapeutic monoclonal antibodies. In this report we have studied the modulation of p185HER2 function with 2 monoclonal antibodies, termed 4D5 and 6E9, which bind the extracellular domain of p185HER2. 4D5 inhibited proliferation of p185HER2 overexpressing SK-BR-3 human breast carcinoma cells (ED50 of approximately 1 nM) but did not inhibit proliferation of cultured human breast carcinoma MCF7 cells, low expressors of p185HER2. Monoclonal antibody 6E9 does not inhibit the growth of either cell line. Antibody binding studies revealed 2 populations of p185HER2 molecules on SK-BR-3 cells: one of high abundance (approximately 2 x 10(6) sites/cell) recognized by 4D5 (Kd approximately 6 nM) and the other of low abundance (2 x 10(4) sites/cell) recognized by 6E9 (Kd approximately 0.1 nM). 4D5, in an agonistic manner, downregulated SK-BR-3 cell surface p185HER2, was internalized, and stimulated p185HER2 phosphorylation in intact cells. Phosphoamino acid analysis of p185HER2 derived from SK-BR-3 cells incubated with the 4D5 monoclonal antibody demonstrated increased tyrosine, serine and threonine phosphorylation. 4D5, on short term (5 min) exposure to SK-BR-3 cells, stimulated inositol lipid hydrolysis as evidenced by increased intracellular levels of inositol polyphosphates (InsP) and sn-1,2-diacylglycerol (sn-1,2-DAG). On longer (24 h) exposure to the cells, the antibody appeared to downregulate this signalling pathway since the intracellular levels of InsP and sn-1,2-DAG decreased by 30 to 40%. 6E9 did not inhibit SK-BR-3 cell proliferation, downregulate surface p185HER2, stimulate receptor phosphorylation, or stimulate the second messenger pathway. Despite these agonistic properties, 4D5 was an inhibitor of SK-BR-3 cell proliferation at all concentrations tested (0.7 to 70 pM). The data suggest that 4D5 is a partial or weak agonist and thus may inhibit cell proliferation by mimicking ligand-like receptor downregulation.

The relationship between detrimental (cachectic) and beneficial (antitumor) effects of tumor necrosis factor (TNF) was studied in mice bearing murine tumors transfected to secrete human TNF. In vitro, the TNF-producing transfectants were resistant to the secreted TNF and grew at rates similar to those of untransfected cells or transfected cells that did not secrete TNF. However, tumors formed by the TNF-secreting cells in vivo remained much smaller than the nonsecreting (transfected and untransfected) tumors. This inhibition of tumor growth required only relatively low serum levels of TNF, persisted for many weeks, and was independent of T cells since it occurred in nude mice. Growth of the TNF-secreting tumors increased dramatically after treatment with anti-human TNF antibody, indicating that extracellular TNF secreted by the tumor cells was necessary for the tumor inhibition. Severe weight loss characteristic of cachexia only occurred in animals with very high serum TNF levels (250 pg/ml) and could be prevented or reversed by anti-TNF antibody treatment. These data are consistent with the existence of a therapeutic window in which persistent exposure to human TNF can lead to prolonged inhibition of tumor growth in the absence of T-cell immunity or severe weight loss and without development of resistant tumor variants.

The HER2 protooncogene encodes a 185-kDa transmembrane protein (p185HER2) with extensive homology to the epidermal growth factor (EGF) receptor. Clinical and experimental evidence supports a role for overexpression of the HER2 protooncogene in the progression of human breast, ovarian, and non-small cell lung carcinoma. These data also support the hypothesis that p185HER2 present on the surface of overexpressing tumor cells may be a good target for receptor-targeted therapeutics. The anti-p185HER2 murine monoclonal antibody (muMAb) 4D5 is one of over 100 monoclonals that was derived following immunization of mice with cells overexpressing p185HER2. The monoclonal antibody is directed at the extracellular (ligand binding) domain of this receptor tyrosine kinase and presumably has its effect as a result of modulating receptor function. In vitro assays have shown that muMAb 4D5 can specifically inhibit the growth of tumor cells only when they overexpress the HER2 protooncogene. MuMAb 4D5 has also been shown to enhance the TNF-alpha sensitivity of breast tumor cells that overexpress this protooncogene. Relevant to its clinical application, muMAb 4D5 may enhance the sensitivity of p185HER2-overexpressing tumor cells to cisplatin, a chemotherapeutic drug often used in the treatment of ovarian cancer. In vivo assays with a nude mouse model have shown that the monoclonal antibody can localize at the tumor site and can inhibit the growth of human tumor xenografts which overexpress p185HER2. Modulation of p185HER2 activity by muMAb 4D5 can therefore reverse many of the properties associated with tumor progression mediated by this putative growth factor receptor. Together with the demonstrated activity of muMAb 4D5 in nude mouse models, these results support the clinical application of muMAb 4D5 for therapy of human cancers characterized by the overexpression of p185HER2.

Activins, dimers of inhibin beta subunits, are potent stimulators of FSH secretion in vivo and in vitro and of FSH beta mRNA expression in rat anterior pituitary cultures. In this study, we investigated the possibility that locally secreted activin B (beta B beta B) may function as an autocrine modulator of basal FSH secretion and expression based on the previous observation that beta B is expressed within gonadotropes. The incubation of cultured rat anterior pituitary cells with a m mouse monoclonal antibody specific for the activin B homodimer (MAb-activin B) significantly attenuated the basal secretion of FSH in a concentration- and time-dependent manner, without influencing LH secretion. Moreover, MAb-activin B selectively inhibited FSH beta mRNA accumulation without affecting either LH beta or alpha subunit mRNAs. The MAb-activin B completely blocked the stimulation of FSH secretion by exogenous activin B, but not by activin A, confirming its specificity. As previously shown, inhibin A and follistatin significantly suppressed basal FSH secretion in these cultures. This inhibitory effect, albeit of lower magnitude, was still evident even in the presence of the MAb-activin B which by itself suppressed basal FSH secretion. These data suggest that the secretion of activin B by the gonadotropes of the anterior pituitary may serve as an autocrine signal in the selective modulation of FSH expression and secretion. Furthermore, the inhibitory actions of inhibins and follistatins on gonadotropes may, in part, be explained by their ability to interfere with the actions of endogenous activin B.

TGF-beta is a potent regulator of immune functions both in vitro and in vivo. The majority of studies have examined changes in immune functions after the addition of TGF-beta that had been previously activated by acid treatment. Peripheral blood mononuclear cells (PBMC) and tumour cells can each produce latent TGF-beta. The role of endogenously produced latent TGF-beta as an autocrine or paracrine regulator of immune functions has not been extensively studied. Monoclonal antibody (mAb) 4A11 was used to detect and neutralize the activity of endogenous TGF-beta 1 produced during lymphocyte activation. We demonstrate that PBMC, after stimulation with interleukin 2 or phytohaemagglutinin-P/12-O-tetradecanoylphorbol 13-acetate, secrete significant quantities of latent TGF-beta 1. Addition of neutralizing mAbs specific for TGF-beta 1 enhances the proliferative response of the PBMC. CHO cell lines engineered to produce latent TGF-beta 1 were poor stimulators of cytotoxic T lymphocyte generation in vitro and significantly suppress natural killer cell activity in nu/nu mice. We conclude that mechanisms exist in vitro and in vivo to convert latent TGF-beta into an active form which can then regulate immune functions in an autocrine/paracrine manner. The possible role of latent TGF-beta produced by tumour cells in immune surveillance is discussed.

The proto-oncogene HER2/neu encodes a protein tyrosine kinase (p185HER2) that is homologous to the human epidermal growth factor receptor. Amplification and/or overexpression of HER2/neu occurs in multiple human malignancies and appears to be integrally involved in progression of some breast and ovarian cancers. Because of this fact, HER2/neu is an intriguing target for specific cancer therapeutic strategies. One such strategy is active specific immunotherapy, in which the immune system is targeted at specific antigens expressed by tumor cells. We have employed a transfected cell line that secretes the extracellular domain of p185HER2 as a source of HER2-derived immunogen in a guinea pig model. The immunized animals developed a cellular immune response, as monitored by delayed-type hypersensitivity, and antisera derived from immunized animals specifically inhibited the in vitro growth of human breast tumor cells overexpressing p185HER2. These data provide support for an immunotherapeutic approach to cancers characterized by overexpression of the HER2/neu proto-oncogene.

We describe the production and characterization of three mAb to transforming growth factor-beta (TGF-beta) and the use of two of them for the development of a TGF-beta 1-specific ELISA and for the study of the regulation of immune function in vitro. All three mAb bound recombinant human TGF-beta 1 (rHuTGF-beta 1) with high affinity and recognized the dimer form of this molecule in immunoblots. mAb 2G7 immunoprecipitated rHuTGF-beta 1, TGF-beta 2, and rHuTGF-beta 3 and neutralized the growth inhibitory activity of all three molecules in vitro on mink lung epithelial-like cells, Mv1Lu, indicating a shared neutralization epitope. mAb 4A11 neutralized and immunoprecipitated only rHuTGF-beta 1, and mAb 12H5 immunoprecipitated rHuTGF-beta 1 but had no effect on the bioactivity of either rHuTGF-beta 1, TGF-beta 2, or rHuTGF-beta 3. These results suggest that a second neutralization epitope may be unique to TGF-beta 1. The ELISA was developed with mAb 4A11 and 12H5, with a range of 0.63 to 40 ng/ml, i.e., a sensitivity of 0.63 ng/ml or 63 pg/sample. The assay is accurate, precise, and specific for the active but not the inactive or latent TGF-beta 1 complex and fails to react with TGF-beta 2, rHuTGF-beta 3, inhibin A, and activin A. Supernatants obtained from serum-free cultures of human PBMC from multiple donors contained significant quantities of TGF-beta 1 (3 to 15 ng/ml), which was detected in the ELISA only after pH 2 treatment to convert latent TGF-beta to the active form. Treatment of the PBMC with either recombinant human IL-2 (rHuIL-2) or PHA-P/PMA enhanced the production of latent TGF-beta 1. mAb 4A11 and 2G7, but not mAb 12H5 enhanced both the proliferative response of PBMC to rHuIL-2/rHuTNF-alpha and PHA-P and the development of the rHuIL-2/rHuTNF-alpha treated PBMC into LAK cells with cytotoxic activity against COLO target cells. These findings suggest that although PBMC secrete latent TGF-beta 1, mechanisms that convert the latent TGF-beta complex into an active form exist in vitro and that the endogenously produced TGF-beta can regulate immune functions in an autocrine fashion.

Infection of mononuclear cells by human immunodeficiency virus (HIV) begins with binding of the viral envelope glycoprotein, gp120, to its receptor, CD4. CD4 contains four extracellular immunoglobulin-like domains, the first of which (V1) is sufficient for HIV binding. V1 contains three sequences homologous to the antigen-complementarity-determining regions (CDR1 to -3) of immunoglobulin variable domains. While all three immunoglobulin CDRs are involved in antigen binding, only amino acids within and flanking the CDR2-like region of CD4 have been shown previously to be involved in gp120 binding. To investigate whether other regions in V1 take part in gp120 binding, we substituted alanine for each of 64 amino acids, including all of the hydrophilic residues in this domain. Mutations at four locations outside the CDR2-like sequence (amino acids 29, 59-64, 77-81, and 85) markedly affected gp120 binding, but not the overall structure of V1 as probed with eight conformationally sensitive monoclonal antibodies. Thus, the gp120-binding site of CD4 is not limited to the CDR2-like sequence and consists of several discontinuous segments. Several amino acids were identified that are critical for the conformation of V1; the importance of these residues suggests some differences in the folding of this domain compared to immunoglobulin variable domains. Three amino acid substitutions were found that increase the affinity for gp120 significantly (1.7- to 2-fold individually and 4.2-fold when combined), suggesting that it may be possible to improve the HIV-blocking ability of CD4-based molecules by increasing their gp120 binding affinity.

A novel relaxin sensitive cell line of apparent smooth muscle origin has been established from a newborn rhesus monkey uterus (NRMU). NRMU cells respond to relaxin, in the presence of 1 microM forskolin, by producing intracellular adenosine 3', 5'-cyclic monophosphate (cAMP). The increase in cAMP levels is dose, time and cell density dependent, reaching peak levels at 10 min when cells are seeded at 1 X 10(5) cells/well. Specificity was demonstrated by neutralization of the relaxin activity with anti-relaxin monoclonal and polyclonal antibodies, degradation of cAMP in the presence of phosphodiesterase, and confirmation of the absence of cGMP. Three synthetic analogs of human relaxin generated a dose-related cAMP response as did synthetic native human relaxin. Natural relaxin purified from human corpora lutea tissue also generated a response similar to synthetic human relaxin. Porcine and rat relaxins also increased levels of cAMP. Insulin, but not IGF I or IGF II, was capable of increasing cAMP levels in NRMU cells, however, 200 ng/mL were required to achieve cAMP levels comparable to 6.25 ng/ml relaxin. Combinations of relaxin with insulin, IGF I or IGF II did not increase cAMP levels above levels obtained with relaxin alone. The effect on NRMU cells of other hormones, growth factors and drugs potentially present in cell culture systems or serum samples was evaluated. In combination with relaxin, oxytocin significantly decreased the cAMP production below the levels induced by relaxin alone, whereas progesterone and prostaglandin E2 resulted in additive increases in cAMP. These data suggest that the NRMU cell line is an appropriate target tissue for studying relaxin-mediated biological responses in vitro as well as functioning as the primary component of a relaxin in vitro bioassay.

We describe a mammalian cell expression system used to rapidly produce microgram quantities of a membrane protein used as an immunogen. A fusion protein expression vector was constructed which contained the signal sequence and 27 amino acids of the Herpes simplex virus glycoprotein D (gD), followed by a factor VIII (fVIII) thrombin cleavage site and the mature tissue factor (TF) sequence. This fusion protein was transiently expressed and then purified using an antibody to gD. The purified fusion protein, gDTF, was incubated with thrombin to remove the gD-fVIII moiety and the resulting rTF served as antigen for the generation of TF-specific antibodies. The antibodies produced were then used for a comparison of the turnover rates of the constitutively and transiently produced fusion protein. In addition, sensitivity to glycosidases indicated that the transiently and constitutively produced recombinant proteins do not contain identical carbohydrate structures.

The human homolog of the rat neu oncogene, HER2 (also termed c-erbB2) has been demonstrated in amplified form in human breast tumors with poor prognosis. Although amplification of the gene correlates with expression of a 185-kDa transmembrane glycoprotein, no extensive information is available regarding the extent of tissue and tumor specificity of this gene product. We have addressed this issue by immunohistochemically evaluating the expression of p185 HER2 in normal tissue and various tumors using monoclonal antibodies (MAbs) to distinct epitopes of its extracellular domain. No detectable levels of p185 HER2 were found in fetal tissues analyzed, with the exception of renal tubules in 2 out of 3 specimens tested and in intestinal epithelium. In adult tissues, detectable levels of this glycoprotein were found in a restricted number of cell types, the expression being heterogeneous among individuals and cell histotypes. Among the neoplasms assayed p185 HER2 was expressed in 46% of primary breast cancers, in 28% of ovarian tumors and in 30% of colon rectum malignancies. No male breast adenocarcinomas were p185-positive. A large number of other tumors tested revealed only a low incidence of expression of the p185. In metastatic breast tumors p185 HER2 was demonstrated homogeneously among multiple autologous lesions and almost invariably (80%) the expression of p185 in the primary lesion correlated with that of the deriving metastases. Our findings indicate that the expression of the p185 HER2 represents a tumor marker of clinical relevance in breast cancer. Whether this holds true for other malignancies remains to be explored.

High levels of expression of either the epidermal growth factor receptor or the receptor-like HER2/neu gene product p185HER2 have been observed in a variety of human malignancies. Because of the association of this high level expression with certain human tumors, we have generated a panel of monoclonal antibodies specific for either the epidermal growth factor receptor or p185HER2 to study their structure, function, and antigenic domains in the normal and neoplastic states. We used the epidermoid carcinoma line A431 to generate five monoclonal antibodies which immunoprecipitate the epidermal growth factor receptor. These monoclonal antibodies bind to the extracellular domain of the epidermal growth factor receptor and demonstrate variable effects on epidermal growth factor binding. We used a stably transfected NIH 3T3 cell line expressing the HER2/neu gene to produce and characterize 10 monoclonal antibodies which immunoprecipitate p185HER2. These monoclonal antibodies bind to the extracellular domain of p185HER2 and do not cross-react with the epidermal growth factor receptor. The characteristics and potential applications of these monoclonal antibodies will be discussed.

We generated a panel of 10 murine monoclonal antibodies (MAbs) that recognize human complement fragment C5a. These MAbs were characterized for their ability to immunoprecipitate 125I-labeled C5a, bind C5a in solid-phase enzyme immunoassay, and block 125I-labeled C5a binding to polymorphonuclear leukocytes. Four of these MAbs had affinity constants for C5a in the 1 X 10(9) to 3 X 10(9) M-1 range. These MAbs blocked C5a-induced neutrophil polarization and chemiluminescence. They blocked the ability of passively administered C5a to cause neutropenia in rabbits. These anti-C5a neutralizing MAbs may have potential therapeutic use in states of complement activation.

A panel of four monoclonal antibodies (MAbs) was generated against recombinant human tumor necrosis factor-alpha (rTNF). These MAbs immunoprecipitate 125I-labeled rTNF, block binding of 125I-labeled rTNF to L929 mouse fibroblasts, and neutralize in vitro cytotoxicity of rTNF and native TNF (nTNF) in the L929 cytotoxicity assay. They define distinct epitopes closely associated with the receptor binding site of rTNF. In Western analysis they bind to both monomeric and dimeric rTNF. Two MAbs recognizing distinct epitopes were used to develop a 'sandwich' enzyme immunometric assay (EIMA) to measure rTNF levels in human serum and other fluids.

A murine immunoglobulin G1 monoclonal antibody was produced that binds to a protease-sensitive, periodate-insensitive epitope on a 43,000-molecular-weight Mycoplasma pneumoniae membrane polypeptide. The 43,000-molecular-weight polypeptide appeared to be a major antigenic component of M. pneumoniae, as determined by immunoblot analysis. This monoclonal antibody reacted with 33 different clinical isolates of M. pneumoniae, but not with normal-flora Mycoplasma species or 18 other microorganisms potentially inhabiting the normal or diseased human respiratory tract. This apparent species-specific monoclonal antibody may have application for the detection of M. pneumoniae antigen in clinical specimens.

Definition of HLA-DR epitopes has been attempted by utilizing monoclonal antibody probes. Hybridoma antibodies L203 and L227, known to bind different epitopes on human Ia-like molecules, were tested for their ability to block cytotoxicity of monoclonal and allogeneic anti-DR antibodies. Monoclonal cytotoxic antibodies segregated into two groups: those more effectively blocked by L203, and those more effectively blocked by L227. Alloantisera also segregated into two groups, but according to their DR specificity. Anti-DR1, -2, and -3 alloantisera were effectively blocked by both L203 and L227, whereas anti-DR7, -w9, -w10, and MT1 alloantisera were not blocked by either. Blocking was not correlated with immunoglobulin class of the alloantibody and further definition of the mechanism of cytotoxicity blocking remains to be elucidated. Based on these data and prior binding and immunochemical studies with L203 and L227, a model is proposed in which the tertiary structure of each DR molecule, or complex of associated molecules on the cell surface, has two reference epitopes, one defined by L203, and another defined by L227. HLA-DR epitopes defined by the cytotoxic monoclonal or alloantibodies to the L203 or L227 epitope in order to begin epitope mapping or grouping.

The HER2/c-erbB-2 gene encodes the epidermal growth factor receptorlike human homolog of the rat neu oncogene. Amplification of this gene in primary breast carcinomas has been show to correlate with poor clinical prognosis for certain cancer patients. We show here that a monoclonal antibody directed against the extracellular domain of p185HER2 specifically inhibits the growth of breast tumor-derived cell lines overexpressing the HER2/c-erbB-2 gene product and prevents HER2/c-erbB-2-transformed NIH 3T3 cells from forming colonies in soft agar. Furthermore, resistance to the cytotoxic effect of tumor necrosis factor alpha, which has been shown to be a consequence of HER2/c-erbB-2 overexpression, is significantly reduced in the presence of this antibody.

A new modification of an HLA-DR typing technique is described which makes DR typing as rapid and simple as routine HLA-A,B,C typing. In this new method, designated the TM1 technique, carboxyfluoresceindiacetate labeled peripheral blood lymphocytes are added directly to DR typing trays. The T cells are then lysed by addition of TM1, a pan-T cytotoxic IgM monoclonal antibody, and residual B-cell reactivity with cytotoxic DR alloantibodies is read as in routine fluorochromasia microlymphocytotoxicity. HLA-DR typing by the TM1 technique compares favorably to typing by methods using B cells enriched by sheep red blood cell rosetting or by Degalan bead columns. The TM1 technique also works well with cells that have been cryopreserved as well as with cells that have been separated from whole blood drawn as much as 3 days earlier. Finally, because TM1 is so effective in lysing normal T lymphocytes, this antibody may prove useful in functional in vitro and in vivo studies requiring T-cell depletion.