In order to study the antigenic phenotype of different hemopoietic cells, we used a series of monoclonal antibodies to investigate normal bone marrow in a standard immunofluorescence assay. The antibodies detected the following antigens: HLA-ABC, beta 2-microglobulin (beta 2m), HLA-DR (Ia), a lymphocyte subset and specific antigen (T and B) HuLy-m2, m3, T lymphocyte antigen (HuLy-m1), lymphocyte T200 antigen (HuLy-m4), a viral-associated antigen (HuLy-m5), and platelet-specific glycoproteins IIb-IIIa (HuPl-m1). The following results were obtained: (a) normoblasts were weakly HLA-ABC+, beta 2m+ and Ia-; all other lymphocyte and platelet antigens were not detected. (b) Myeloid cells at all stages of differentiation (promyelocytes, myelocytes, metamyelocytes, and neutrophils) were HLA-ABC+; beta 2m+; HuLy-m1-, m2-, m3+/- (20%), m4+, m5+/- (20%); HuPl-m1-; in addition, promyelocytes and myelocytes were Ia+ but neutrophils and metamyelocytes were Ia-. (c) Lymphocytes were HLA-ABC+, beta 2m+, Ia+/- (20-30%), HuLy-m1+/- (40-50%), m2+/- (60-70%), m3+, m4+, m5+; Pl-m1-. (d) Platelets and megakaryocytes were HLA-ABC+; beta 2m+; Ia-; HuLy-m1+-, m2-, m3-, m4-, m5-, HuPl-m1+, and the putative "megakaryocyte precursors" were HuPl-m1+, Ia-, HuLy-m1-. The different cell types in bone marrow could readily be distinguished, particularly cells of the myeloid series (Ia and HuLy-m4, m5), lymphocytes (Ia and HuLy-m1, m2, m3), and platelets and their precursor cells (HuPl-m1). This simple method of defining cellular phenotypes in bone marrow has demonstrated the practicality of using monoclonal antibodies to identify marrow cells and should be of diagnostic value.
Human tumor necrosis-alpha (hTNF-alpha) was chemically conjugated to the murine anti-Ly-2.1 T cell antibody using heterobifunctional crosslinking agents SAMSA and SPDP. SDS-PAGE analysis of the affinity purified conjugate consisted mainly of 1:1 and 1:2 (Ly-2.1:TNF) complexes. Conjugated hTNF retained 50% of its cytotoxic activity by the L929 cytolytic assay, with an IC50 = 0.12 ng/ml. hTNF-Ly-2.1 was also cytotoxic to E3 cells (Ly-2.1+ve) with an IC50 = 1.7 microg/ml - 3 times more cytotoxic to these cells than non-conjugated hTNF in vitro. However in vivo hTNF-Ly-2.1 conjugates were more toxic to mice than hTNF. In vivo blood clearance studies in E3 tumor bearing CBF1 mice demonstrated that the half life of the conjugate was 2 hr, compared to 20 min for hTNF. In biodistribution studies, tumor accumulation of 3% was seen for hTNF-Ly-2.1 while for unconjugated hTNF no activity in tumor was detected 24hr post injection. A single dose of hTNF-Ly-2.1 increased the accumulation of 125I-anti-Ly-2.1 by 3 fold compared to controls. However, the antitumor effect of hTNF-Ly-2.1 on E3 cells in vivo was marginal with some tumor growth retardation at day 1-3. The results of these in vitro and in vivo studies on chemically conjugated h-TNF-MoAb will be helpful in the design of novel recombinant fusion proteins for targeting the biologic activity of TNF to tumours.
Summary Several approaches were made to produce new monoclonal antibodies (MoAb) to human colonic tumours using different immunogens, such as colon cell lines, and tissue and serum from patients with colon cancer. Six MoAb were produced but all were found lo be reactive with carcino‐embryonic antigen (CEA). Through tissue reactivity and competitive inhibition studies it was found that four epitopes could be detected: CEA‐I detected by MoAb I‐l and O‐l; CEA‐2 detected by MoAb i7C4; CEA‐3 detected by MoAb LK‐4 and XPX‐13 and CEA‐4 detected by MoAb JGT‐13. CEA‐2 was also found on granulocytes and probably detects normal cross‐reacting antigen (NCA). In formalin‐fixed tissues the CEA‐specific epitopes were absent from normal adult tissues, but this was an artefact of the fixation procedure and fresh, non‐fixed colonic tissues were clearly reactive. All six MoAb reacted with > 70% of colonic tumours when tested by the immunoperoxidase technique and gave different patterns of reactivity reflecting the differential expression of the CEA 1–4 epitopes. Despite the apparent specificity of these MoAb for colon cancer, serum testing using MoAb gave similar results to CEA polyclonal antibodies, that is the MoAb gave no obvious advantage. Thus efforts to produce new reagents to colon cancer yielded only anti‐CEA monoclonal reagents. Clearly, other new approaches are required to produce better diagnostic reagents for this common disease.
503 Background & Aims: The importance of the galactose-α(1,3)galactose (Galα1,3Gal) epitope to xenograft rejection is well-known. Because mice have a functional α1,3-galactosyltransferase gene and express Galα1,3Gal, they do not have anti-Galα1,3Gal antibodies. In contrast, Gal k/o mice produced by homologous disruption of α1,3-galactosyltransferase gene have preformed anti-Galα1,3Gal antibodies. Using Gal k/o mice, we have generated hybridomas producing monoclonal antibodies to Galα1,3Gal by immunization with Galα1,3Gal and characterized the genetic control of anti-Galα1,3Gal antibody binding activity in the Gal k/o mice. Methods: Total RNA was extracted from the hybridomas derived from Gal k/o mice immunized with lymphocytes and thymocytes from Gal(+) mice. RT-PCR was performed to amplify the cDNA of hypervariable region of immunoglobulin heavy chain (VH). The PCR products were subcloned with pCR 2.1 TA cloning vectors (Invitrogen, Carlsbad, CA). and then sequenced by Auto sequencer. Results: Hypervariable regions of heavy chains of three IgM, two IgG1, and one IgG3 antibodies from six hybridomas were cloned and sequenced. Four of six monoclonals, three IgM and one IgG1, used a closely-related groups of VH genes to encode their antigen binding activity. The V-segments of these four VH genes were very similar (97-100% nucleic acid, and 94-100% amino acid sequence identity) to the mouse germline gene VH441. A fifth antibody expressed an IgG3 VH gene was very similar (97% nucleic acid, and 94% amino acid sequence identity) to the mouse germline gene VH11. One IgG1 antibody expressed a VH gene different from the others (55-57% nucleic acid, and 37-41% amino acid sequence identity) and most closely-related to the mouse J558 gene family. The monoclonal antibody (12.15) exhibiting the highest binding affinity for Galα1,3Gal was encoded by the germline gene VH441 (100% nucleic acid sequence identity). Conclusions: These data suggest that mouse germline gene VH441 is important for encoding antibody binding to the Galα1,3Gal epitope in mice. Comparison of the VH441 gene with human VH germline genes has established that the VH3-11 gene, one of the genes that encode human antibody responses to pig hepatocytes, is most closely-related to the mouse VH441 gene family. This data suggests that the genes encoding antibody responses to the same xenograft target antigen share extensive evolutionary conservation between distantly related species.
Humans with breast cancer have T-cells in their lymph nodes which recognize a peptide sequence within the variable number of tandem repeats of the mammary mucin, MUC1, which is overexpressed in breast cancer. To find means of making this recognition event into a potent immune response to breast cancer, we used a murine tumor model and have examined the parameters of the immune response to human mucin (MUC1) expressed in murine BALB/c 3T3 cells. We then sought to boost this response with MUC1-containing synthetic peptides, fusion proteins, and natural mucin (HMFG). MUC1+3T3 cells were found to be rejected by BALB/c mice by day 15 due to a cellular [CD3+, Ly2+ (CD8+)] response. The cellular rejection response was accompanied by the generation of CD8+ cytotoxic T-cells, CD4+ delayed-type hypersensitivity, and little anti-MUC1 antibody. This immune response is presumably of the TH1 type (which occurs in CD8 as well as CD4 cells) of CD8+ cytotoxic cells. By contrast, mice immunized with the MUC1 synthetic peptide, a fusion protein, or HMFG have good antibody responses, a delayed-type hypersensitivity reaction, but no cytotoxic T-cells and less tumor protection, possibly a TH2 type response. We conclude that CD8+ cytotoxic anti-mucin cells can produce significant antitumor responses in vivo to a human "tumor" antigen expressed in murine cells; immunization with soluble synthetic or native materials leads to the "humoral" (TH2) type of immunity, and efforts need to be made to convert this to a TH1-type response.
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