Semirational design of a potent, artificial agonist of fibroblast growth factor receptors.

Fibroblast growth factors (FGFs) are being investigated in human clinical trials as treatments for angina, claudication, and stroke. We designed a molecule structurally unrelated to all FGFs, which potently mimicked basic FGF activity, by combining domains that (1) bind FGF receptors (2) bind heparin, and (3) mediate dimerization. A 26-residue peptide identified by phage display specifically bound FGF receptor (FGFR) 1c extracellular domain but had no homology with FGFs. When fused with the c-jun leucine zipper domain, which binds heparin and forms homodimers, the polypeptide specifically reproduced the mitogenic and morphogenic activities of basic FGF with similar potency (EC50 = 240 pM). The polypeptide required interaction with heparin for activity, demonstrating the importance of heparin for FGFR activation even with designed ligands structurally unrelated to FGF. Our results demonstrate the feasibility of engineering potent artificial agonists for the receptor tyrosine kinases, and have important implications for the design of nonpeptidic ligands for FGF receptors. Furthermore, artificial FGFR agonists may be useful alternatives to FGF in the treatment of ischemic vascular disease.

Urokinase receptor antagonists: discovery and application to in vivo models of tumor growth.

Urokinase receptor antagonists based on the growth factor domains of both human and murine urokinase which show sub-nanomolar affinities for their homologous receptors have been expressed as recombinant proteins. Further modification of these molecules by preparing fusions with the constant region of human IgG has led to molecules with high affinities and long in vivo half-lives. Smaller peptidic inhibitors have been obtained by a combination of bacteriophage display and peptide analog synthesis. All of these molecules inhibit the binding of the growth factor domain of uPA to the uPA receptor and enhance binding of the uPA receptor to vitronectin. Protein uPA receptor antagonists were tested in an in vivo tumor model using the human breast carcinoma MDAmb231 in immunodeficient mice. Both human and murine receptor antagonists showed significant inhibition of primary tumor growth, demonstrating that in vivo, both tumor and stromal cell uPA receptor dependent plasminogen activation can modulate tumor growth.

Urokinase receptor antagonists inhibit angiogenesis and primary tumor growth in syngeneic mice.

Urokinase plasminogen activator (uPA) and its receptor are key components of a cell surface proteolytic cascade used by tumor cells and capillary endothelial cells for basement membrane invasion, a process required for metastasis and angiogenesis. We have cloned, expressed, and purified the epidermal growth factor-like domain of murine uPA alone and fused it to the Fc portion of human IgG as high-affinity murine urokinase receptor antagonists. These molecules are potent inhibitors of murine urokinase binding to its receptor and inhibit angiogenesis in an in vitro model of capillary tube formation in fibrin gels. In vivo, basic fibroblast growth factor-induced neovascularization and B16 melanoma growth in syngeneic mice are also substantially suppressed by these molecules. Coupled with previous studies showing inhibition of metastasis, these findings suggest that urokinase receptor antagonists may be useful therapeutically as inhibitors of tumor progression.

Stimulation of cloned human T lymphocytes via the CD3 or CD28 molecules induces enhancement in vascular endothelial permeability to macromolecules with participation of type-1 and type-2 intercellular adhesion pathways.

Perivascular accumulation of CD29+CD45R0+ memory T lymphocytes at sites of chronic inflammation such as rheumatoid synovium is commonly associated with the localized increase in the endothelial permeability. We have recently demonstrated that a direct interaction between activated CD29+CD45R0+ memory T lymphocytes and vascular endothelial cells (EC) results in the increased permeability of EC. In this report, we have investigated effects on antigen-specific T cell receptor (TcR) alpha/beta+ human T lymphocyte clones on the endothelial permeability to albumin. Our results show that CD29+CD45R0+ cloned human T lymphocytes augment endothelial permeability by a noncytolytic process requiring surface contact between T lymphocytes and EC. Both cytolytic and noncytolytic cloned T lymphocytes were capable of augmenting endothelial permeability and this process did not involve active lysis of EC. Stimulation of T lymphocytes via the CD3/TcR or CD28 molecules resulted in significant enhancement in the ability of T lymphocytes to influence endothelial permeability. Pretreatment of T lymphocytes with monoclonal antibodies directed at either CD11a/CD18 (LFA-1) or CD2 molecules or that of EC with monoclonal antibodies directed at either CD54 (ICAM-1) or CD58 (LFA-3) molecules significantly inhibited T lymphocyte-induced enhancement in endothelial permeability, thus indicating that activated T lymphocytes utilize both type-1 (CD11a/CD18CD54) and type-2 (CD2CD58) intercellular adhesion pathways to augment endothelial permeability and signals received via CD3 or CD28 molecules on T lymphocytes further enhance this process. Furthermore, proinflammatory cytokines interleukin 1 and tumor necrosis factor but not proinflammatory cytokines interleukin 1 and tumor necrosis factor but not interleukin 6 induced resistance in EC to T lymphocyte-mediated effects on their permeability. Collectively, these observations may provide insights into molecular mechanism(s) underlying pathophysiology of localized chronic inflammatory responses in general and more specifically selective accumulation of chronically activated memory T lymphocytes at sites of chronic inflammation.

Ability of human T lymphocytes to adhere to vascular endothelial cells and to augment endothelial permeability to macromolecules is linked to their state of post-thymic maturation.

The accumulation of mononuclear cells at sites of chronic inflammation is dependent on a number of factors including localized adherence of lymphocytes to vascular endothelial cells (EC), cytokine-mediated increased adhesiveness of endothelium, chemotactic factors and endothelial permeability. The present study investigates two of the above attributes of lymphocyte-EC interaction: namely, the ability of maturationally distinct subpopulations of human T lymphocytes to adhere to vascular EC and to increase vascular endothelial permeability to macromolecules in an in vitro model. Thus, human T lymphocytes were separated into CD4+ CD8-helper/inducer, CD4- CD8+ cytotoxic/suppressor, CD29+ CD45RA- CD45RO+ memory, and CD29- CD45RA+ CD45RO- naive/virgin T subpopulations, were activated with PHA and PMA, and then examined for their adherence to EC and also for their effect on endothelial permeability. Upon activation, cells within each of the above four subpopulations exhibited increased adherence to EC. In contrast, resting CD29+ CD45RA- CD45RO+ memory T lymphocytes exhibited two to three times greater ability to adhere to EC than their CD29- CD45RA+ CD45RO- naive/virgin counterparts. Consistent with their increased adherence to EC, CD29+ CD45RO+ memory T lymphocytes, when activated, significantly increased endothelial permeability to albumin. Although activated CD45RA+ naive T lymphocytes exhibited increased adherence to EC, these cells failed to increase significantly endothelial permeability. Similar to their polyclonal counterparts, Ag-specific CD4+ CD29+ CD45RO+ T cell clones, but not their actively released mediators, also increased endothelial permeability via a noncytolytic mechanism(s). This ability of CD29+ CD45RO+ memory T lymphocytes to augment endothelial permeability may facilitate their transendothelial migration into extravascular space. These observations may provide additional insights into molecular mechanism(s) underlying pathophysiology of localized chronic inflammatory responses in general and more specifically selective accumulation of CD29+/CD45RO+ memory T lymphocytes at sites of chronic inflammation such as rheumatoid synovium.

Stimulation via the CD3 and CD28 molecules induces responsiveness to IL-4 in CD4+CD29+CD45R- memory T lymphocytes.

Although both IL-2 and IL-4 can promote the growth of activated T cells, IL-4 appears to selectively promote the growth of those helper/inducer and cytolytic T cells which have been activated via their CD3/TCR complex. The present study examines the participation of CD28 and certain other T cell-surface molecules in inducing T cell responsiveness to IL-4. Purified small high density T cells were cultured in the absence of accessory cells with various soluble anti-human T cell mAb with or without soluble anti-CD3 mAb and their responsiveness to IL-4 was studied. None of the soluble anti-T cell mAb alone was able to induce T cell proliferation in response to IL-4. A combination of soluble anti-CD3 with anti-CD28 mAb but not with mAb directed at the CD2, CD5, CD7, CD11a/CD18, or class I MHC molecules induced T cell proliferation in response to IL-4. Anti-CD2 and anti-CD5 mAb enhanced and anti-CD18 mAb inhibited this anti-CD3 + anti-CD28 mAb-induced T cell response to IL-4. In addition, anti-CD2 in combination with anti-CD3 and anti-CD28 mAb induced modest levels of T cell proliferation even in the absence of exogenous cytokines. IL-1, IL-6, and TNF were each unable to replace either anti-CD3 or anti-CD28 mAb in the induction of T cell responsiveness to IL-4, but both IL-1 and TNF enhanced this response. The anti-CD3 + anti-CD28 mAb-induced response to IL-4 was exhibited only by cells within the CD4+CD29+CD45R- memory T subpopulation, and not by CD8+ or CD4+CD45R+ naive T cells. When individually cross-linked with goat anti-mouse IgG antibody immobilized on plastic surface, only anti-CD3 and anti-CD28 mAb were able to induce T cell proliferation. These results indicate that the CD3 and CD28 molecules play a crucial role in inducing T cell responsiveness to IL-4 and that the CD2, CD5, and CD11a/CD18 molecules influence this process.

IL-2-activated human killer lymphocytes but not their secreted products mediate increase in albumin flux across cultured endothelial monolayers. Implications for vascular leak syndrome.

When cultured with IL-2, human lymphoid cells acquire the ability to lyse various NK-resistant tumor targets. Due to their anti-tumor cytolytic effect, clinical trials with IL-2 alone or IL-2 + IL-2-activated killer (IAK) lymphocytes have been undertaken. However, infusion of therapeutically effective doses of IL-2 is associated with the development of systemic toxicity characterized by exaggerated endothelial permeability, also known as vascular leak syndrome. The present study was designed to examine the effects of IAK cells and their secreted products on vascular endothelial permeability by using an in vitro endothelial permeability model in which the flux of FITC-albumin across endothelial cell (EC) monolayers was measured. When endothelial monolayers were exposed to IAK cells for 2 h, significant increases in the transendothelial permeability to albumin were observed. Exposure of EC to lymphocytes cultured in the absence of IL-2 did not induce significant alteration in the endothelial permeability. In addition, neither culture supernatants of IAK cells nor purified recombinant cytokines, including IL-1 beta, IL-2, IL-3, IL-4, IL-6, TNF-alpha, GM-CSF, M-CSF, and IFN-gamma, had any effect on endothelial permeability in this model. Prior activation of EC with TNF-alpha did not alter the increased permeability induced by IAK cells or lack of it by nonactivated lymphocytes. Dexamethasone treatment of IAK cells abolished their anti-tumor cytolytic effect but only partially inhibited their ability to induce increased endothelial permeability. Pretreatment of IAK cells with mAb directed at the CD11a/CD18 (LFA-1) adhesion complex, and that of EC with mAb directed at the ICAM-1 molecule, inhibited the IAK cell-induced increase in endothelial permeability. These results demonstrate that direct cell-to-cell contact between IAK cells and EC is necessary and sufficient to cause increased endothelial permeability in this model system, and may therefore be an important factor contributing to the development of the vascular leak syndrome observed clinically.

Distinct regulatory effects of IL-4 and TNF-alpha during CD3-dependent and CD3-independent initiation of human T-cell activation.

The present study examines the effects of IL-4 and TNF-alpha on the CD3-dependent (Ag/MHC-initiated or anti-CD3 mAb-initiated) and CD3-independent (IL-2-initiated) pathways of the initiation of human T-cell activation. Both IL-4 and TNF-alpha significantly augmented the CD3-dependent T-cell proliferation induced by either irradiated OKT3 hybridoma cells or allogeneic B cells. In contrast, the CD3-independent IL-2-initiated T-cell proliferation was enhanced by TNF-alpha and significantly inhibited by IL-4. Although the growth-enhancing effects of both IL-4 and TNF-alpha on the CD3-dependent T-cell proliferation were noticeable regardless of when these cytokines were introduced in culture, the inhibitory effect of IL-4 on the CD3-independent IL-2-initiated T-cell activation was observed only if IL-4 was added at the initiation but not later than 24 hr of "T cells + IL-2" cultures. The growth-enhancing effects of both IL-4 and TNF-alpha on the CD3-dependent T-cell activation were not confined to any one subset of T cells. On the other hand, IL-4 inhibited the IL-2-induced proliferation of CD4+ (helper/inducer) T cells and CD45R+ (virgin) T cells but not that of CD8+ (cytotoxic/suppressor) T cells and CD45R (memory) T cells. When examined for their effects on cytokine production, CD3-dependent production of IL-2 and IFN-gamma was affected by neither cytokine, whereas IL-4 strongly inhibited the production of IFN-gamma by IL-2-stimulated T cells. Consistent with their enhancing and inhibitory effects, respectively, on IL-2-induced T-cell proliferation, TNF-alpha augmented and IL-4 inhibited the development of IL-2-stimulated MHC-unrestricted cytolytic (MUC) T-cell activity directed against tumor cells. When deprived of IL-2, MUC T cells rapidly lose their cytolytic activity, and despite its inhibitory effect on the development of MUC T cells, exposure of IL-2-deprived MUC T cells with decaying cytolytic activity to IL-4 retards the decay in their cytolytic activity. These results suggest the differential regulatory effects of IL-4 and TNF-alpha during human T-cell activation.

Differential regulatory signals delivered by antibody binding to the CD28 (Tp44) molecule during the activation of human T lymphocytes.

Molecule CD28 (Tp44) is expressed on the surface of majority of human T cells and has been implicated to play an active role in the regulation of T cell growth. The present study examines the effect of antibody binding to the CD28 molecule during T cell activation. Anti-CD28 but not isotype-matched anti-CD5 mAb consistently augmented anti-CD3-induced and IL-2-induced T cell proliferation and subsequent release of soluble CD25 molecule. When added together, mAb anti-CD28 and anti-CD5 acted synergistically to cause 2- to 7-fold enhancement of T cell activation induced by anti-CD3 mAb or IL-2 with no effect on the development of non-MHC-restricted IL-2-activated killer T cells. In contrast, alloantigen-induced T cell proliferation, soluble CD25 release, and the subsequent development of CTL were all inhibited by anti-CD28 mAb. Moreover, alloantigen-induced proliferative response of both CD4+ and CD8+ T cells was inhibited by anti-CD28 without affecting the cytolytic effect of CTL. Because valency of anti-CD28 binding has been implicated as an important factor in signal transduction, this was explored in the allogeneic MLR by using Fab and F(ab')2 fragments of anti-CD28 mAb and anti-mouse kappa mAb. The inhibitory effect of anti-CD28 mAb in the MLR was reversed by cross-linking of anti-CD28 mAb with anti-mouse kappa mAb. In addition, cross-linking of the CD28 molecule on alloactivated T lymphoblasts but not that on resting T cells with anti-CD28 and anti-mouse kappa induced their proliferation in the absence of the priming alloantigen. These results indicate that stimulatory or inhibitory signals delivered through the CD28 molecule are determined by the degree of cross-linking of this molecule. In addition, these results also suggest that Ag-induced CD3-TCR-mediated T cell responses are more dependent on signals delivered through the CD28 molecule than those induced with anti-CD3, and thus these results have implications for potential use of anti-CD28 in sustained propagation of Ag-specific T cells.

Interleukin-2 activated human killer lymphocytes: lack of involvement of interferon in the development of IL-2-activated killer lymphocytes.

When cultured with native or recombinant interleukin-2 (IL-2) human small agranular lymphocytes acquire the ability to kill various tumor targets. The development of these IL-2 activated killer (IAK) cells, also known as LAK, is observed in the absence of antigen or mitogen. Interferons are known to augment the lytic effects of natural killer cells and cytolytic T lymphocytes. Our study was undertaken to examine the effect of human alpha, beta, and gamma interferons on the induction and the effector phase of IAK function. When cultured with small lymphoid cells IFN alone did not induce anti-tumor cytolytic activity in those lymphocytes. Despite their known anti-proliferative effects, none of the 3 IFN species at any concentrations tested inhibited the development of IAK activity when present during the entire culture period. IFN neither inhibited nor augmented the development of IAK cells under suboptimal conditions. Furthermore, activation of IAK cells was not affected by the presence of neutralizing antibodies to either alpha or gamma IFN. Post-activation exposure of IAK cells to IFN also failed to either augment or inhibit their lytic activity. Thus, neither endogenously generated nor exogenously added IFN had any effect on the IAK system, in contrast to their augmenting effects on NK cells and CTL.

Immunoregulatory T lymphocytes in man. Soluble antigen-specific suppressor-inducer T lymphocytes are derived from the CD4+CD45R-p80+ subpopulation.

Regulation of the immune response in man is largely dependent on interactions between cells of the cluster designation 4+ (CD4+) helper/inducer sublineage and the CD8+ suppressor/cytotoxic sublineage. When cultured with autologous antigen-primed CD4+ lymphocytes, CD8+ cells differentiate into suppressor T cells (Ts) that specifically inhibit the response of fresh autologous CD4+ cells to the priming antigen only. The current study was undertaken to analyze the roles in this suppressor circuit of subpopulations of the CD4+ sublineage distinguished from one another on the basis of their binding (or lack of binding) to monoclonal antibodies against molecules p80 (Leu8) and CD45R (p220/Leu18/2H4). When examined for the proliferative responses to alloantigenic stimuli, each of the four: CD4+p80+, CD4+p80-, CD4+CD45R+, and CD4+CD45R- populations proliferated vigorously, synthesized interleukin 2 (IL-2) and interferon and released soluble IL-2 receptors. However, the responses to soluble antigens such as Candida and diphtheria toxoid were exhibited by CD4+CD45R-, CD4+p80+, and CD4+p80- cells, but not by CD4+CD45R+ cells. When examined for their ability to induced CD8+ Ts in the Candida-driven suppressor-induction culture system, only CD4+p80+ and CD4+CD45R- cells induced strong suppression. Further, when CD4+CD45R- cells were separated into CD4+CD45R-p80+ and CD4+CD45R-p80- subpopulations, despite the ability of both subpopulations to respond to Candida, only CD4+CD45R-p80+ cells induced autologous CD8+ Ts. Activated CD8+ Ts suppressed not only proliferation but also the release of soluble IL-2 receptors by autologous antigen-activated CD4+ cells. Thus, the antigen-specific suppressor-inducer T cells appear to be derived from the CD4+CD45R-p80+ (Leu3+, Leu8+, 2H4-) subpopulation of the CD4+ sublineage.

Interleukin 2-activated human lymphocytes exhibit enhanced adhesion to normal vascular endothelial cells and cause their lysis.

When cultured with native or recombinant interleukin 2 (IL 2), human lymphoid cells proliferate and acquire the ability to lyse both NK-sensitive and NK-resistant tumor targets. Such IL 2-activated killer (IAK) cells generally do not destroy nonmalignant nontransformed cells. Due to their apparent specificity for tumor cells, adoptive immunotherapeutic trials of IAK cells and IL 2 have been initiated, with promising results. However, infusion of high doses of IL 2 causes systemic toxicity in patients and experimental animals resulting in the development of a vascular leakage syndrome. Certain aspects of such toxicity suggest IL 2-induced, cell-mediated destruction of normal tissue. This study examines the interaction between IL 2-induced human lymphoid cells and endothelial cells (EC). IL 2, in a dose-dependent manner, causes lymphocytes to strongly adhere to EC, but not to tumor cells, fibroblasts, or epithelial cells. In addition, these IL 2-activated lymphocytes were highly cytotoxic not only to NK-resistant Daudi cells but also to vascular and corneal EC. The IAK cells caused lysis of not only human EC but also bovine EC. Although IAK cells did not display significant adherence to normal human fibroblasts or epithelial cells, when brought together by 50 X G centrifugation, these targets were lysed by IAK cells. The ability to lyse EC was not confined to any single subpopulation of IL 2-activated lymphocytes. The lysis of EC was mediated by both IL 2-activated large granular lymphocytes and small agranular lymphocytes. Furthermore, cells within both CD4+ and CD8+ sublineages of T cells, and also non-T subpopulations, mediated IL 2-induced cytolysis of EC. The destruction of EC by IAK cells may contribute in part to the systemic toxicity associated with infusions of high doses of IL 2.

Biological effects of recombinant human tumor necrosis factor and its novel muteins on tumor and normal cell lines.

We investigated optimal conditions for cytotoxicity to tumor cell lines by recombinant human tumor necrosis factor (rhTNF) and the effect of amino-terminal deletions on the bioactivity of the rhTNF molecule. Two of four deletion muteins (-4 and -7) of rhTNF exhibit 2- to 3-fold enhancement of cytotoxicity/cytostasis against a variety of human carcinomas, a fibrosarcoma, and a melanoma cell line with no toxicity on normal fibroblastic and epithelial cultures. Of the two other muteins the -8 displayed equivalent and/or increased cytotoxicity/cytostasis while the -10 was consistently less cytotoxic than the parent on the same cell lines. Continuous exposure to TNF for greater than or equal to 96 h led to maximal cytotoxicity to tumor lines (99.99% with L929 cells) with no evidence of recovery. Pretreatment with actinomycin D (0.003-10 micrograms/ml for 1 h) rendered 82% of rhTNF-resistant cell lines (both tumor and normal) susceptible to its cytotoxic action within 24 h. However, the highest nontoxic concentrations of Actinomycin D necessary for rendering normal cell lines susceptible to TNF action were about 10-3000-fold higher than those necessary for converting resistant tumor cell lines. Similarly, preinfection of L929 cells with vesicular stomatitis virus (multiplicity of infection, 10(-2)-10(-4) for 1 h) rendered the cells 2-10-fold more susceptible to the cytotoxic action of rhTNF in 18 h. Our data suggest that rhTNF and its muteins represent potentially useful anticancer agents; however, adequate dosing and prolonged exposure may be critical in demonstrating cytotoxicity/cytostasis. The data also show that although normal and tumor cell lines became susceptible to cytotoxicity by rhTNF and actinomycin D, combination therapy of the two agents may be possible at defined concentrations.

Interleukin 2-activated human killer cells are derived from phenotypically heterogeneous precursors.

When cultured with native or recombinant human interleukin 2 (IL 2), human peripheral blood non-adherent mononuclear cells (NAMNC) acquire the ability to lyse both NK-sensitive and NK-resistant tumor target cells. The development of these IL 2-activated killer (IAK) cells, also known as LAK, is observed in the absence of exogenous antigen or mitogen. This study describes the ability of various subpopulations of human peripheral blood NAMNC with defined surface phenotype to generate the IAK activity. Human NAMNC were separated into various subpopulations on the basis of the ability to bind monoclonal antibodies, activated with IL 2, and were examined for the cytolytic effect on various tumor target cells. Although CD16+ (Leu-11+) NK cells from NAMNC could become IAK cells when cultured with IL 2, removal of these cells from NAMNC had no effect on the latter's ability to generate the IAK effect. When CD16- NAMNC were separated into CD2+ E rosette-forming T cells (ERFC) and CD2- non-T (non-ERFC) subpopulations, both subpopulations generated the IAK activity. The ability of monoclonal antibody-defined subpopulations of T and non-T cells to generate IAK cells was then examined. Both CD4+ and CD8+ subsets isolated by either positive or negative selection generated the IAK activity. Similarly, CD20+ (B1+) B cells and CD20- non-T (null) cells developed into IAK cells when cultured with IL 2. In contrast, Leu-7+ T cells failed to generate the IAK activity. CD4+ and CD8+ subsets were additionally separated into narrower subpopulations by using monoclonal antibodies anti-Leu-8 and 9.3 respectively, and were examined for their ability to generate IAK cells. Precursors of IAK cells were derived from each of the four: CD4+, Leu-8+ (inducer), CD4+, Leu-8- (helper/amplifier), CD8+, 9.3+ (cytolytic), and CD8+, 9.3- (suppressor) subpopulations of T cells. Thus, the IAK activity appears to be derived from phenotypically heterogeneous and otherwise functionally diverse human lymphoid cells and is not confined to any single subpopulation.