Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 11 de 11
Filter
Add more filters










Publication year range
1.
Cancer Res ; 61(12): 4750-5, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11406547

ABSTRACT

Antitumor monoclonal antibodies must bind to tumor antigens with high affinity to achieve durable tumor retention. This has spurred efforts to generate high affinity antibodies for use in cancer therapy. However, it has been hypothesized that very high affinity interactions between antibodies and tumor antigens may impair efficient tumor penetration of the monoclonal antibodies and thus diminish effective in vivo targeting (K. Fujimori et al., J. Nucl. Med., 31: 1191-1198, 1990). Here we show that intrinsic affinity properties regulate the quantitative delivery of antitumor single-chain Fv (scFv) molecules to solid tumors and the penetration of scFv from the vasculature into tumor masses. In biodistribution studies examining a series of radioiodinated scFv mutants with affinities ranging from 10(-7)-10(-11) M, quantitative tumor retention did not significantly increase with enhancements in affinity beyond 10(-9) M. Similar distribution patterns were observed when the scFv were evaluated in the absence of renal clearance in anephric mice, indicating that the rapid renal clearance of the scFv was not responsible for these observations. IHC and IF evaluations of tumor sections after the i.v. administration of scFv affinity mutants revealed that the lowest affinity molecule exhibited diffuse tumor staining whereas the highest affinity scFv was primarily retained in the perivascular regions of the tumor. These results indicate that antibody-based molecules with extremely high affinity have impaired tumor penetration properties that must be considered in the design of antibody-based cancer therapies.


Subject(s)
Antibody Affinity , Antigens, Neoplasm/immunology , Immunoglobulin Fragments/immunology , Immunoglobulin Fragments/metabolism , Ovarian Neoplasms/metabolism , Receptor, ErbB-2/immunology , Animals , Antigens, Neoplasm/metabolism , Epitopes/immunology , Epitopes/metabolism , Female , Humans , Kidney/metabolism , Kinetics , Mice , Mice, Inbred Strains , Mice, SCID , Ovarian Neoplasms/blood supply , Ovarian Neoplasms/immunology , Rabbits , Tissue Distribution , Xenograft Model Antitumor Assays
2.
J Immunol ; 166(10): 6112-7, 2001 May 15.
Article in English | MEDLINE | ID: mdl-11342630

ABSTRACT

We tested the hypothesis that bispecific Abs (Bsab) with increased binding affinity for tumor Ags augment retargeted antitumor cytotoxicity. We report that an increase in the affinity of Bsab for the HER2/neu Ag correlates with an increase in the ability of the Bsab to promote retargeted cytotoxicity against HER2/neu-positive cell lines. A series of anti-HER2/neu extracellular domain-directed single-chain Fv fragments (scFv), ranging in affinity for HER2/neu from 10(-7) to 10(-11) M, were fused to the phage display-derived NM3E2 human scFV: NM3E2 associates with the extracellular domain of human FcgammaRIII (CD16). The resulting series of Bsab promoted cytotoxicity of SKOV3 human ovarian carcinoma cells overexpressing HER2/neu by human PBMC preparations containing CD16-positive NK cells. The affinity for HER2/neu clearly influenced the ability of the Bsab to promote cytotoxicity of (51)Cr-labeled SKOV3 cells. Lysis was 6.5% with an anti-HER2/neu K(D) = 1.7 x 10(-7) M, 14.5% with K(D) = 5.7 x 10(-9) M, and 21.3% with K(D) = 1.7 x 10(-10) M at 50:1 E:T ratios. These scFv-based Bsab did not cross-link receptors and induce leukocyte calcium mobilization in the absence of tumor cell engagement. Thus, these novel Bsab structures should not induce the dose-limiting cytokine release syndromes that have been observed in clinical trials with intact IgG BSAB: Additional manipulations in Bsab structure that improve selective tumor retention or facilitate the ability of Bsab to selectively cross-link tumor and effector cells at tumor sites should further improve the utility of this therapeutic strategy.


Subject(s)
Adjuvants, Immunologic/metabolism , Adjuvants, Immunologic/toxicity , Antibodies, Bispecific/metabolism , Antibodies, Bispecific/toxicity , Antibody-Dependent Cell Cytotoxicity , Antigens, Neoplasm/immunology , Antigens, Neoplasm/metabolism , Binding Sites, Antibody , Antibody Specificity , Calcium Signaling/immunology , Cytotoxicity Tests, Immunologic , Humans , Immunoglobulin Variable Region/metabolism , Intracellular Fluid/immunology , Intracellular Fluid/metabolism , Kinetics , Receptor, ErbB-2/immunology , Receptor, ErbB-2/metabolism , Receptors, IgG/immunology , Receptors, IgG/metabolism , Tumor Cells, Cultured
3.
Nucl Med Biol ; 27(4): 339-46, 2000 May.
Article in English | MEDLINE | ID: mdl-10938467

ABSTRACT

Intravenously administered anti-tumor single-chain Fv (scFv) and diabody molecules exhibit rapid clearance kinetics and accumulation in tumors that express their cognate antigen. In an attempt to fit the rate of isotope decay to the timing of delivery and duration of tumor retention, anti-HER2/neu CHX-A" DTPA-C6.5K-A scFv and diabody conjugates were labeled with the alpha-particle emitter (213)Bi (t(1/2) = 47 min). Radioimmunotherapy studies employing 0.64, 0.35, or 0.15 microCi of (213)Bi-labeled C6.5K-A diabody or 1.1, 0.6, or 0. 3 microCi of (213)Bi-labeled C6.5K-A scFv were performed in nude mice bearing early, established SK-OV-3 tumors. Only the 0.3 microCi dose of (213)Bi-labeled C6.5K-A scFv resulted in both acceptable toxicity and a reduction in tumor growth rate. The specificity of the anti-tumor effects was determined by comparing the efficacy of treatment with 0.3 and 0.15 microCi doses of (213)Bi-labeled C6.5K-A scFv and (213)Bi-labeled NM3E2 (an irrelevant scFv) in nude mice bearing large established tumors. The 0.3 microCi dose of (213)Bi on both the C6.5K-A and NM3E2 scFvs resulted in similar anti-tumor effects (p = 0.46) indicating that antigen-specific targeting was not a factor. This suggests that the physical half-life of (213)Bi may be too brief to be effectively paired with systemically-administered diabody or scFv molecules.


Subject(s)
Alpha Particles , Bismuth/therapeutic use , Immunoglobulin Fragments/therapeutic use , Neoplasms, Experimental/radiotherapy , Radioimmunotherapy , Animals , Male , Mice , Mice, Nude
4.
Int J Cancer ; 70(2): 221-9, 1997 Jan 17.
Article in English | MEDLINE | ID: mdl-9009164

ABSTRACT

Geldanamycin belongs to the family of benzoquinoid ansamycin tyrosine-kinase inhibitors. We have examined its effects on Her-2/neu kinase activity, protein expression level, and proliferation of Her-2+ malignant cells. In SK-BR-3 breast-cancer cells, short-time treatment with geldanamycin completely abrogated gp30-ligand-induced activation of Her-2 without a change of receptor-expression level. Longer treatment of intact cells with geldanamycin induced decreased steady-state Her-2 autophosphorylation activity, which correlated with reduction of Her-2 protein expression and phosphotyrosine content of several proteins. The decrease was time- and dose-dependent, starting after 1 hr at 100 nM concentration and reaching completion by 24 hr. The reduction of the Her-2 protein level probably resulted from increased degradation, since the Her-2 mRNA level remained constant. Geldanamycin effects were not specific for Her-2, since the non-receptor tyrosine-kinase fyn was inhibited equally. In contrast to these results, protein-kinase-C activity was not affected. In 3 other malignant cell lines expressing different amounts of Her-2 (SK-BR-3 > SK-OV-3 > OVCAR3 > MCF7), geldanamycin also effectively reduced Her-2-kinase activity proportionally to the decrease of protein expression. In contrast, in a [3H]-thymidine-uptake assay, cell growth was meaningfully inhibited by geldanamycin at nanomolar concentrations only in SK-BR-3 (IC50 2 nM) and MCF7 (IC50 20 nM), while OVCAR3 was only moderately sensitive (IC50 2 microM) and SK-OV-3 was clearly resistant to geldanamycin. In direct comparison with herbimycin A, another benzoquinoid ansamycin that has been more thoroughly characterized, the biologic effects of geldanamycin were more pronounced.


Subject(s)
Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Neoplasm Proteins/antagonists & inhibitors , Quinones/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Adenocarcinoma/pathology , Benzoquinones , Breast Neoplasms/pathology , Cell Division/drug effects , Enzyme Activation , Female , Humans , Lactams, Macrocyclic , Ligands , Ovarian Neoplasms/pathology , Protein-Tyrosine Kinases/antagonists & inhibitors , RNA, Messenger/biosynthesis , RNA, Neoplasm/biosynthesis , Rifabutin/analogs & derivatives , Tumor Cells, Cultured
5.
Cancer Res ; 54(16): 4362-70, 1994 Aug 15.
Article in English | MEDLINE | ID: mdl-8044783

ABSTRACT

The efficacy, specificity, and toxicity of bismuth (212Bi) alpha particle-mediated radioimmunotherapy was evaluated in nude mice bearing a murine lymphoma transfected with the human CD25 [human Tac; interleukin 2 receptor alpha (IL-2R alpha)] gene. The therapeutic agent used was the tumor-specific humanized monoclonal antibody anti-Tac conjugated to 212Bi. The human IL-2R alpha-expressing cell line was produced by transfecting the gene encoding human Tac into the murine plasmacytoma cell line SP2/0. The resulting cell line, SP2/Tac, expressed approximately 18,000 human IL-2R alpha molecules/cell. Following s.c. or i.p. injection of 2 x 10(6) SP2/Tac cells into nude mice, rapidly growing tumors developed in all animals after a mean of 10 and 13 days, respectively. The bifunctional chelate cyclohexyldiethylenetriaminepentaacetic acid was used to couple 212Bi to the humanized anti-Tac monoclonal antibody. This immunoconjugate was shown to be stable in vivo. Specifically, in pharmacokinetic studies in nude mice, the blood clearance patterns of i.v. administered 205/206Bi-anti-Tac and coinjected 125I-anti-Tac were comparable. The toxicity and therapeutic efficacy of 212Bi-anti-Tac were evaluated in nude mouse ascites or solid tumor models wherein SP2/Tac cells were administered either i.p. or s.c., respectively. The i.p. administration of 212Bi-anti-Tac, 3 days following i.p. tumor inoculation, led to a dose-dependent, significant prolongation of tumor-free survival. Doses of 150 or 200 microCi prevented tumor occurrence in 75% (95% confidence interval, 41-93%) of the animals. In the second model, i.v. treatment with 212Bi-anti-Tac 3 days following s.c. tumor inoculation also resulted in a prolongation of the period before tumor development. However, prevention of tumor occurrence decreased to 30% (95% confidence interval, 11-60%). In both the i.p. and s.c. tumor trials, 212Bi-anti-Tac was significantly more effective for i.p. (P2 = 0.0128 50/100 microCi 212Bi-anti-Tac versus 50/100 microCi Mik beta; P2 = 0.0142 150/200 microCi anti-Tac versus 150/200 microCi Mik beta) and for s.c. tumors (P2 = 0.0018 100 microCi anti-Tac versus 100 microCi Mik beta; P2 = 0.0042 200 microCi anti-Tac versus 200 microCi Mik beta 1) than the control antibody Mik beta 1 coupled to 212Bi at comparable dose levels. In contrast to the efficacy observed in the adjuvant setting, therapy of large, established s.c. SP-2/Tac-expressing tumors with i.v. administered 212Bi-anti-Tac (at doses up to 200 microCi/animal) failed to induce tumor regression.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Antibodies, Monoclonal/therapeutic use , Bismuth/therapeutic use , Leukemia-Lymphoma, Adult T-Cell/radiotherapy , Radioimmunotherapy/methods , Radioisotopes/therapeutic use , Receptors, Interleukin-2/immunology , Animals , Antibodies, Monoclonal/adverse effects , Antibodies, Monoclonal/metabolism , Bismuth/adverse effects , Bismuth/metabolism , Dose-Response Relationship, Immunologic , Drug Screening Assays, Antitumor , Humans , Leukemia-Lymphoma, Adult T-Cell/immunology , Leukemia-Lymphoma, Adult T-Cell/metabolism , Mice , Mice, Nude , Radioimmunotherapy/adverse effects , Radioisotopes/adverse effects , Radioisotopes/metabolism , Radiotherapy Dosage , Receptors, Interleukin-2/metabolism , Tissue Distribution
6.
J Biol Chem ; 268(28): 20725-8, 1993 Oct 05.
Article in English | MEDLINE | ID: mdl-7691802

ABSTRACT

Bacterial lipopolysaccharide (LPS) induces a pleiotropic activation of the immune system which might subsequently result in septic shock. One of the cell surface receptors for LPS is the glycophosphatidylinositol-anchored protein CD14. Binding of LPS to CD14 induces production of lymphokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), IL-6, and IL-8, and CD14 is subsequently released from the cell surface. However, the mechanism of signaling via CD14 is still not known. We report here that protein tyrosine kinase (PTK) p56lyn is coupled to the LPS receptor CD14 in human monocytes. LPS rapidly activates CD14-associated p56lyn simultaneously with PTKs p58hck and p59c-fgr. Inhibition of PTKs by herbimycin A completely blocks LPS-induced down-modulation of CD14 and production of TNF-alpha and IL-1. These data suggest a critical role of PTKs in the LPS/CD14-mediated signal transduction pathway in human monocytes.


Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Lipopolysaccharides/pharmacology , Protein-Tyrosine Kinases/metabolism , src-Family Kinases , Humans , In Vitro Techniques , Lipopolysaccharide Receptors , Lymphokines/biosynthesis , Monocytes/metabolism , Phosphorylation , Precipitin Tests
7.
Proc Natl Acad Sci U S A ; 88(5): 1996-2000, 1991 Mar 01.
Article in English | MEDLINE | ID: mdl-2000405

ABSTRACT

Addition of interleukin 2 (IL-2) to IL-2-dependent T cells results in tyrosine protein kinase signal transduction events even though the IL-2 receptor alpha and beta chains lack intrinsic enzymatic activity. Here we report that addition of IL-2 to IL-2-dependent human T cells transiently stimulates the specific activity of p56lck, a member of the src family of nonreceptor tyrosine protein kinases expressed at high levels in T lymphocytes. The ability of IL-2 to induce p56lck activation was found to be independent of the capacity of p56lck to associate with either CD4 or CD8. Following IL-2 treatment, p56lck was found to undergo serine/threonine phosphorylation modifications that resulted in altered mobility of the lck gene product on polyacrylamide gels. These observations raise the possibility that p56lck participates in IL-2-mediated signal transduction events in T cells.


Subject(s)
Interleukin-2/pharmacology , Protein-Tyrosine Kinases/metabolism , Signal Transduction , T-Lymphocytes/immunology , CD4 Antigens/analysis , Cell Line , Clone Cells , Enzyme Activation , Humans , Kinetics , Lymphocyte Specific Protein Tyrosine Kinase p56(lck) , T-Lymphocytes/drug effects , T-Lymphocytes/enzymology
8.
Nature ; 348(6301): 557-60, 1990 Dec 06.
Article in English | MEDLINE | ID: mdl-1701034

ABSTRACT

The T lymphocyte surface protein CD4 is an integral membrane glycoprotein noncovalently associated with the tyrosine protein kinase p56lck. In normal T cells, surface association of CD4 molecules with other CD4 molecules or other T-cell surface proteins, such as the T-cell antigen receptor, stimulates the activity of the p56lck tyrosine kinase, resulting in the phosphorylation of various cellular proteins at tyrosine residues. Thus, the signal transduction in T cells generated through the surface engagement of CD4 is similar to that observed for the class of growth factor receptors possessing endogenous tyrosine kinase activity. As CD4 is also the cellular receptor for the human immunodeficiency virus (HIV), binding of the virus or gp120 (the virus surface protein responsible for specific CD4+ T-cell association) could mimic the types of immunological interactions that have previously been found to stimulate p56lck and trigger T-cell activation pathways. We have evaluated this possibility and report here that binding of HIV-1 or the virus glycoprotein gp120 to CD4+ human T cells fails to elicit detectable p56lck-dependent tyrosine kinase activation and signalling, alterations in the composition of cellular phosphotyrosine-containing proteins, or changes in intracellular Ca2+ concentration.


Subject(s)
CD4 Antigens/physiology , CD4-Positive T-Lymphocytes/physiology , Calcium/physiology , HIV Envelope Protein gp120/physiology , HIV-1/immunology , Protein-Tyrosine Kinases/physiology , Antibodies, Monoclonal , Enzyme Activation , Humans , In Vitro Techniques , Molecular Weight , Phosphotyrosine , Receptor Aggregation , Recombinant Proteins/metabolism , Signal Transduction , Time Factors , Tyrosine/analogs & derivatives , Tyrosine/metabolism
9.
Nature ; 338(6212): 257-9, 1989 Mar 16.
Article in English | MEDLINE | ID: mdl-2784195

ABSTRACT

The CD4 T-cell surface antigen is an integral membrane glycoprotein of relative molecular mass 55,000 which binds class II major histocompatibility complex (MHC) molecules expressed on antigen presenting cells (APCs). It is thought to stabilize physical interactions between T cells and APCs (for a review, see ref. 1). Evidence is accumulating that suggests that CD4 can transduce an independent signal during T-cell activation. It has recently been shown that CD4 expressed on human and murine T cells is physically associated with the Src-related tyrosine protein kinase p56lck (refs 7, 8). These results indicate that CD4 can function as a signal transducer and suggest that tyrosine phosphorylation events may be important in CD4-mediated signalling. Here, we present evidence that cross-linking of the CD4 receptor induces a rapid increase in the tyrosine-specific protein kinase activity of p56lck and is associated with the rapid phosphorylation of one of the subunits (zeta) of the T-cell receptor complex on tyrosine residues. These data provide direct evidence for a specific CD4 signal transduction pathway that is mediated through p56lck and suggest that some of the tyrosine phosphorylation events detected during antigen-mediated T-cell activation may result from signalling through this surface molecule.


Subject(s)
Protein-Tyrosine Kinases/metabolism , Receptors, Virus/metabolism , Signal Transduction , Animals , Mice , Receptors, HIV , T-Lymphocytes/metabolism
10.
Cell ; 55(2): 301-8, 1988 Oct 21.
Article in English | MEDLINE | ID: mdl-3262426

ABSTRACT

The CD4 and CD8 T cell antigens are thought to transduce an independent signal during the process of T cell activation. We report our evaluation of the possible involvement of the lymphocyte-specific tyrosine kinase p56lck in these transduction pathways. Our data demonstrate that p56lck is specifically modulated with either CD4 or CD8 following antibody-mediated cross-linking of these molecules and that a large fraction of the total cellular lck protein can be coimmunoprecipitated with these surface glycoproteins. These results suggest that p56lck is functionally and physically associated with CD4/CD8 in normal murine T lymphocytes and support the concept that an independent signal is transduced by the interaction of these surface molecules with major histocompatibility complex determinants.


Subject(s)
Antigens, Differentiation, T-Lymphocyte/analysis , Protein-Tyrosine Kinases/analysis , Signal Transduction , Animals , Cell Line , Major Histocompatibility Complex , Mice , T-Lymphocytes/analysis
11.
Mol Cell Biol ; 8(10): 4353-61, 1988 Oct.
Article in English | MEDLINE | ID: mdl-3141789

ABSTRACT

The lymphocyte-specific tyrosine protein kinase p56lck is abundantly expressed in L3T4+ (CD4+) and Lyt-2+ (CD8+) T-lymphocytes, where it is predominantly phosphorylated in vivo on the carboxy-terminal tyrosine residue 505 (Y-505). Upon exposure to activating signals (mitogenic lectins, antibodies to the T-cell receptor), the p56lck expressed in normal cloned murine T-cells is modified into a product which migrates at approximately 59 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels and which possesses several amino-terminal serine phosphorylations. The changes in both mobility and amino-terminal phosphorylation can be reproduced by known activators of protein kinase C (4 alpha-phorbol 12 beta-myristate, dioctanoylglycerol), suggesting that this signal transduction pathway (or related pathways) mediates at least part of these events. Interestingly, agents raising intracellular calcium (such as A23187) cause the appearance of several of these amino-terminal phosphorylation changes but do not cause the pronounced shift in electrophoretic mobility. These data suggest that at least two serine kinase systems are implicated in the alterations of p56lck associated with T-cell activation and that the lck gene product plays a critical role in normal T-cell physiology.


Subject(s)
Lymphocyte Activation , Protein-Tyrosine Kinases/metabolism , T-Lymphocytes/enzymology , Animals , Calcimycin/pharmacology , Concanavalin A/pharmacology , Lymphocyte Specific Protein Tyrosine Kinase p56(lck) , Mice , Peptide Mapping , Phosphoproteins/metabolism , Phosphorylation , T-Lymphocytes/classification , Tetradecanoylphorbol Acetate/pharmacology
SELECTION OF CITATIONS
SEARCH DETAIL
...