HuM291, a humanized anti-CD3 antibody, is immunosuppressive to T cells while exhibiting reduced mitogenicity in vitro.

BACKGROUND: OKT3, a mouse monoclonal antibody (Ab) specific for the human CD3 complex on T cells, is a potent immunosuppressive agent used for the treatment of acute allograft rejection. The utility of the drug has been limited by a neutralizing anti-mouse Ab response and adverse side effects resulting from T cell activation and systemic cytokine release. T cell activation is caused by OKT3-mediated cross-linking of T cells and Fc receptor-bearing cells. Studies in the mouse model have shown that global T cell activation is not necessary for immunosuppression, as Fc receptor-nonbinding anti-CD3 Abs can suppress graft rejection in the absence of the activation effects seen with Fc receptor-binding Abs. Thus, a humanized anti-CD3 antibody with a low affinity for Fc receptors might improve immunosuppressive therapy by reducing the side effects associated with OKT3. METHODS: We developed a mouse monoclonal Ab, M291, which competes with OKT3 for binding to T cells. Humanized, complementary-determining region-grafted versions of M291 featuring various Fc were engineered, including a previously described IgG2 mutant deficient in Fc receptor binding (HuM291). RESULTS: Compared with OKT3 and HuM291-IgG1, HuM291 was significantly less mitogenic to T cells in vitro and induced the release of much lower levels of the cytokines tumor necrosis factor-alpha, interferon-gamma, and interleukin-10. Despite this reduction in T cell activation, HuM291 retained the ability to modulate the CD3 complex and inhibit the mixed lymphocyte reaction. CONCLUSIONS: When evaluated in vivo, HuM291 may be an immunosuppressive agent associated with less of the acute toxicity and immunogenicity seen with OKT3 therapy.

Anti-CD3-based bispecific antibody designed for therapy of human B-cell malignancy can induce T-cell activation by antigen-dependent and antigen-independent mechanisms.

Anti-CD3 x anti-B-cell antigen bispecific monoclonal antibodies (bsAbs) can redirect T-cell-mediated lysis toward malignant B cells. Clinical trials with CD3-based bsAbs have shown toxicity in patients which is likely related to nonspecific T-cell activation and targeting. Our current studies were designed to explore the mechanisms responsible for the observed in vivo toxicity by evaluating the immunologic effects of 2 different bsAb preparations in vitro. 1D10 was used as the tumor specific arm of the bsAbs. This antibody reacts with a variant of HLA-DR found on a majority of pre-B- and B-cell malignancies, and normal B cells in some individuals. Anti-CD3 served as the T-cell specific arm. A 1D10 x anti-CD3 bispecific IgG (bsIgG) produced using the hybrid-hybridoma method was compared to a 1D10 x anti-CD3 bispecific F(ab')2 [bsF(ab')2] produced using the leucine zipper technique. In cytotoxicity assays, both bsIgG and bsF(ab')2 induced lysis by pre-activated T cells of 1D10 (+) malignant B cells. bsIgG at high concentrations also induced lysis of 1D10 (-) tumor cells, while bsF(ab')2 did not. Proliferation of T cells induced by bsIgG and bsF(ab')2 was also evaluated. Both forms of bsAbs induced T-cell proliferation in the presence of antigen (+) Raji cells, while only bsIgG did so in the presence of antigen (-) malignant B cells. bsF(ab')2 induced T-cell activation in the absence of any tumor cells when testing was performed on samples where the 1D10 target antigen was present on normal peripheral blood B cells. We conclude that non-specific T-cell activation from bsAbs can occur in an antigen-independent manner due to the Fc/Fc receptor (FcR) interaction, or in an antigen-dependent manner when antigen is expressed on normal or tumor cells. Both mechanisms may have been responsible for the toxicity observed in prior clinical studies.

CD3 ligation on immature thymocytes generates antagonist-like signals appropriate for CD8 lineage commitment, independently of T cell receptor specificity.

The signals that direct differentiation of T cells to the CD4 or CD8 lineages in the thymus remain poorly understood. Although it has been relatively easy to direct differentiation of CD4 single positive (CD4+) cells using combinations of antibodies and pharmacological agents that mimic receptor engagements, equivalent stimuli do not induce efficient maturation of CD8+ cells. Here we report that, irrespective of the MHC-restriction specificity of the TCR, differentiation of mature CD8+ thymocytes can be induced by ligation of CD3 polypeptides on immature thymocytes with a F(ab')2 reagent (CD3fos-F(ab')2). The tyrosine phosphorylation patterns stimulated by CD3fos-F(ab')2 have been shown to resemble those delivered to mature T cells by antagonist peptides, which are known to direct positive selection of CD8+ cells, and we can show that this reagent exhibits potent antagonistic-like activity for primary T cell responses. Our results suggest a distinction in the signals that specify lineage commitment in the thymus. We present a model of thymocyte differentiation that proposes that the relative balance of signals delivered by TCR engagement and by p56lck activation is responsible for directing commitment to the CD8 or CD4 lineages.

Human IgG2 variants of chimeric anti-CD3 are nonmitogenic to T cells.

The mouse anti-human CD3 mAb OKT3 is a potent immunosuppressive agent used for the treatment of acute transplant rejection. OKT3 therapy is associated with acute toxicity resulting from in vivo T cell activation and systemic cytokine release, and a human anti-mouse Ab response. T cell activation is thought to be triggered by CD3 cross-linking mediated by the Abs bridging T cells and Fc receptor-bearing cells. Recent studies in a mouse model indicate that anti-mouse CD3 Abs with low affinity for Fc receptors can achieve immunosuppression without T cell activation, toxicity, or an anti-Ab response. To obtain an analogous Ab to improve the current anti-human CD3 therapy, a humanized Ab with low affinity for Fc receptors is needed. In this study, we introduced mutations into the upper CH2 region of IgG2 and expressed the altered Fc as chimeric OKT3 Abs. Compared with chimeric OKT3 IgG1, IgG2, IgG3, and IgG4, the IgG2 mutants were less mitogenic to T cells, and they did not induce the release of TNF-alpha, IFN-gamma, or IL-2. In parallel, we observed no functional interaction of the IgG2 mutant Abs with K562 cells, which express the IgG2-binding Fc receptor on their surface. Despite no measurable T cell activation, the mutant Abs could still modulate the CD3 complex. When coupled to a humanized anti-CD3, the IgG2 variant may provide a drug with less acute toxicity and immunogenicity, but may still retain potent immunosuppressive properties.

Signals through CD8 or CD4 can induce commitment to the CD4 lineage in the thymus.

Differentiation of thymocytes into mature single-positive T cells is an ordered process involving sequential interactions between T cell receptor (TCR), co-receptors (CD4 or CD8) and their appropriate major histocompatibility complex-encoded ligands. Precisely how these receptor/co-receptor engagements determine lineage commitment is still controversial, but recently it has been suggested that quantitative differences in the signal transmitted by co-ligation of CD4 versus CD8 with TCR might provide the discriminating signal. We examine this hypothesis, using bispecific F(ab')2 antibodies to mimic TCR/ co-receptor engagement during thymocyte differentiation. These bispecific antibodies lack Fc and can engage surface molecules without extensive cross-linking or targeting to Fc receptor-bearing cells. We show that TCR/CD3 co-ligation with CD4 induces efficient differentiation of mature CD4 lineage cells, irrespective of their TCR specificity. Interestingly, TCR/CD3 co-ligation with CD8 also induces maturation of CD4 T cells, although less efficiently, but not of CD8 T cells. Thus, although the signals delivered by co-ligation of TCR and CD8 appear weaker than from co-ligation of TCR and CD4, the outcome from either engagement is the same. These data suggest that differences in signal intensity alone do not determine lineage commitment in the thymus, but that distinct signals are required for CD4 and CD8 single-positive cell differentiation.

Nonmitogenic anti-CD3 monoclonal antibodies deliver a partial T cell receptor signal and induce clonal anergy.

Anti-CD3 monoclonal antibodies (mAbs) are potent immunosuppressive agents used in clinical transplantation. However, the activation-related adverse side effects associated with these mAbs have prompted the development of less toxic nonmitogenic anti-CD3 mAb therapies. At present, the functional and biochemical consequences of T cell exposure to nonmitogenic anti-CD3 is unclear. In this study, we have examined the early signaling events triggered by a nonmitogenic anti-CD3 mAb. Like the mitogenic anti-CD3 mAb, nonnmitogenic anti-CD3 triggered changes in the T cell receptor (TCR) complex, including zeta chain tyrosine phosphorylation and ZAP-70 association. However, unlike the mitogenic anti-CD3 stimulation, nonmitogenic anti-CD3 was ineffective at inducing the highly phosphorylated form of zeta (p23) and tyrosine phosphorylation of the associated ZAP-70 tyrosine kinase. This proximal signaling deficiency correlated with minimal phospholipase Cgamma-1 phosphorylation and failure to mobilize detectable Ca2+. Not only did biochemical signals delivered by nonmitogenic anti-CD3 resemble altered peptide ligand signaling, but exposure of Th1 clones to nonmitogenic anti-CD3 also resulted in functional anergy. Finally, a bispecific anti-CD3 X anti-CD4 F(ab)'2 reconstituted early signal transduction events and induced proliferation, suggesting that defective association of lck with the TCR complex may underlie the observed signaling differences between the mitogenic and nonmitogenic anti-CD3.

A bispecific antibody prolongs survival in mice bearing lung metastases of syngeneic mammary adenocarcinoma.

In the present study we tested whether T cells retargeted with a bispecific antibody (bsAb) could block the growth of lung metastases of syngeneic mammary adenocarcinoma in immunocompetent mice. BALB/c mice were injected i.v. with tumor and i.p. with a genetically engineered bispecific F(ab')2 [bs(Fab')2] having specificity for murine CD3 epsilon chain and for the gp52 mouse mammary tumor viral glycoprotein, which is expressed on the tumor cells. The bs(Fab')2 was physically stable in blood and serum, was removed from the body with a half-time of 12-15 h, and accumulated in lymphoid tissue where it bound to T cells. We show that treatment of tumor bearing mice with the bs(Fab')2 significantly prolonged their survival relative to untreated controls. Two other genetically engineered bs(Fab')2s having specificity for murine CD3 epsilon chain and irrelevant antigens did not inhibit tumor growth. In addition, survival was not affected by bsAb therapy using a variant tumor cell line that expressed low levels of the gp52 target antigen. Inhibition of tumor growth was even more evident by histologic analysis. Treatment with the relevant bs(Fab')2 resulted in a marked reduction of tumor burden in lung sections taken on days 7, 9 and 11. This is the first report demonstrating that a bsAb can inhibit the growth of syngeneic solid tumor metastases in mice without addition of T cell activators.

Bispecific antibodies retarget murine T cell cytotoxicity against syngeneic breast cancer in vitro and in vivo.

Bispecific antibodies with specificity for CD3 and a tumor antigen can redirect cytolytic T cells to kill tumor targets, regardless of their natural specificity. To assess the clinical potential of bispecific antibodies for treatment of human cancers we have, in the present study, adapted a totally synergeic mouse model to the targeting of mouse T cells against mouse tumors in immunocompetent mice. We show that gp52 of the mouse mammary tumor virus (MTV) can serve as a tumor-specific antigen for redirected cellular cytotoxicity. Chemically crosslinked and genetically engineered bispecific antibodies with specificities for gp52 and murine CD3 epsilon-chain induced activated mouse T cells to specifically lyse mouse mammary tumor cells from cultured lines and primary tumors from C3H-MTV+ mice. Retargeted T cells also blocked the growth of mammary tumors in vitro as well as their growth in syngeneic mice. These findings identify murine MTV-induced mammary adenocarcinomas as a solid-tumor, animal model for retargeting T cells with bispecific antibodies against syngeneic breast cancer.

NGF mRNA is not decreased in frontal cortex from Alzheimer's disease patients.

Alzheimer's disease (AD) is characterized by neuronal dysfunction and degeneration in certain brain regions such as cortex, hippocampus and basal forebrain. Specific neurochemical defects such as decreases in cholinergic enzymes and in the amounts of mRNA in AD brain have also been reported. Nerve growth factor (NGF), a protein necessary for the development, regulation and survival of basal forebrain cholinergic neurons (BFCN), is synthesized in target areas of BFCN (cortex, hippocampus) and is supplied to BFCN by retrograde transport. Thus, NGF is under investigation both as a potential therapeutic agent and for its possible involvement in the pathogenesis of AD. In this study, postmortem brain tissues from both control and AD cases were investigated for amounts of poly (A)+ mRNA and NGF mRNA in the frontal cortex, a region rich in cholinergic afferents. Yields of poly(A)+ mRNA were similar from normal and AD tissues. Human NGF mRNA comigrated with murine NGF mRNA on Northern blots. Additionally, dot blot quantitation demonstrated that NGF mRNA levels do not differ in the inferior frontal gyrus of normal and AD patients. Thus, we conclude that levels of mRNA in general, and of NGF mRNA in particular, are unchanged in the frontal cortex of individuals affected by AD.

The role of T cell activation in anti-CD3 x antitumor bispecific antibody therapy.

Anti-CD3 x antitumor bispecific Ab can retarget T cell mediated lysis in an MHC-independent fashion and prevent tumor growth in animal models. Two bispecific Ab preparations that differ in the presence or absence of Fc were compared in the 38C13 immunocompetent murine lymphoma model to evaluate how functional Fc and T cell activation impact on response to bispecific Ab therapy. Bispecific (bs) IgG contained functional Fc and was purified from hybrid-hybridoma Ab product. Bsf(ab')2 lacked functional Fc, and was genetically constructed using the leucine zipper technique. In vitro, bsF(ab')2 induced tumor cell lysis by activated T cells more effectively than bsIgG. However, bsF(ab')2 failed to induce T cell activation in the absence of tumor cells, and did so more slowly than bsIgG when tumor cells were present. In vivo, bsIgG induced nonspecific T cell activation whereas bsF(ab')2 did not. In therapy experiments, bsIgG inhibited tumor growth in mice although a single dose of bsF(ab')2 had minimal antitumor effect. BsF(ab')2 was capable of preventing tumor growth and improving survival when mice were also treated with T cell activators (IL-2 or staphylococcal enterotoxin B), or given repeated bsF(ab')2 doses. We conclude that therapeutic response to bispecific Ab was not dependent on functional Fc, but did require T cell activation. The use of bifunctional constructs that lack functional Fc therefore allows for separate manipulation of T cell retargeting and T cell activation and deserves further evaluation as a potential immunotherapy for malignancy.

Construction of a human Ig combinatorial library from genomic V segments and synthetic CDR3 fragments.

A naive combinatorial Ig library was constructed from semi-synthetic V genes consisting of human genomic V segments and synthetic CDR3 fragments. VH and V kappa segments were amplified from human genomic DNA by polymerase chain reaction using V subgroup-specific primers. The amplified VH and V kappa segments were combined with synthetic oligonucleotides containing a J region and CDR3 with amino acid sequence variations, resulting in complete V genes. These V genes were cloned into a phagemid expression vector in a single-chain form fused to the carboxyl-terminus of the M13 minor coat protein III. Phagemid particles displaying the single chain hybrid proteins on their surface were screened with Con A as Ag. Several clones showing specific binding to Con A were obtained after four rounds of selection and were further analyzed for their binding properties and DNA sequences. This method provides a novel way to create a naive combinatorial library without using mRNA from B lymphocytes as template. The method should be useful to isolate human antibodies that react with self-Ag.

Formation of a bispecific antibody by the use of leucine zippers.

A new method is described for the production of bispecific F(ab')2 heterodimers using leucine zippers. Two heterodimer-forming "zipper" peptides derived from the Fos and Jun proteins were respectively linked to the Fab' portions of two different mAb by gene fusion. The antibodies used were 145-2C11, which binds to murine CD3, and anti-Tac, which binds to the p55 chain of the human IL-2R. Anti-Tac Fab'-Jun and anti-CD3 Fab'-Fos were expressed individually as F(ab'-zipper)2 homodimers in the mouse myeloma cell line Sp2/0. When these homodimers were reduced at the hinge region to form monomers and then reoxidized together, the resulting end products were mostly F(ab'-zipper)2 heterodimers. Bispecific anti-CD3 x anti-Tac F(ab'-zipper)2 heterodimers produced by this method were shown to be highly effective in recruiting cytotoxic T cells to lyse IL-2R-bearing HuT-102 cells in vitro.

Coumarin necrosis--a review of the literature.

Skin and soft tissue necrosis is a rare complication of anticoagulation therapy. Two patients who exhibited the spectrum of this disorder are described. The clinical features, etiology, pathophysiology, and treatment of coumarin skin necrosis are outlined, and the English-language literature is reviewed.

The integration of spina bifida children--are their needs being met?

After eleven years of vigorously promoting integration, the majority of Sheffield Children's Hospital spina bifida patients are now successfully integrated. A parallel study in Greater Manchester confirmed that parents, teachers and the spina bifida children are overwhelmingly in favour of integration. A large number of children were transferred from special to ordinary schools, and very few had to be transferred back to segregated schooling. The parents found substantial social and educational advantages in integration, together with good levels of involvement with their child's education. The most important areas in need of greater funding were considered to be: non-teaching support for toiletting, visiting specialists, particularly physiotherapists, and in-service education of teachers.

Malignant transformation of BALB/3T3 cells by residue organic mixtures from drinking water.

Malignant transformation of BALB/3T3 1-13 cells results following treatment with complex mixtures of residue organic chemicals from drinking water samples of five U. S. cities. The U. S. Environmental Protection Agency provided the residues which were obtained by liquid-liquid solvent extraction, and sorption-desorption on XAD-2 resin of samples concentrated by reverse osmosis from drinking water of Philadelphia, PA; New Orleans, LA; Miami, FL; Seattle, WA; and Ottumwa, IA. Transformation was observed using a focus formation assay following 72-hr exposure of cells to residues from one or more samples from each city. Malignancy of several transformed and normal clones was evaluated in athymic (nu/nu) mice. The usefulness of such a transformation assay in detecting potential carcinogenic compounds contained in complex mixtures and the evaluation of possible health effects of residue organics in drinking water are discussed.

Clinical evaluation of the minitek differential system for identification of Enterobacteriaceae.

Forty-one stock organisms and 581 fresh clinical isolates were used in comparing the Minitek system to conventional tubed media to determine if this system is feasible and accurate for a high-volume clinical microbiology laboratory. In addition to comparison with tubed media, the following parameters were tested: (i) reproducibility of disks, (ii) the effect of variation in inoculum size, (iii) the effect of the age of culture, and (iv) the effect of predispensing disks. A total of 5,947 disks were compared with tube reactions; 95.9% agreed on the initial reading. After repeating questionable tube and disk reactions, this agreement rose to 98.2%. Using the disk reactions and the identification schema currently in use with tubed media, 94.9% of the Minitek final identifications agreed with those of tubed media; 1.5% gave differing identifications and 3.6% were inconclusive. This system is accurate, inexpensive, flexible, and convenient.