A humanized anti-CD3 antibody, HuM291, with low mitogenic activity, mediates complete and reversible T-cell depletion in chimpanzees.

BACKGROUND: An anti-CD3 antibody that reduces cytokine release syndrome (CRS) while maintaining immunosuppression would be a major advance in the treatment of acute allograft rejection. A humanized (Hu) anti-CD3 IgG2 Ab, HuM291 gamma2 M3 (HuM291; Protein Design Labs, Inc., Mountain View, CA), was engineered with mutations in the upper CH2 region of the Fc domain. The mutations were intended to reduce affinity for Fcgamma receptors, thought to be relevant to CRS. METHODS: In vitro studies using chimpanzee peripheral blood mononuclear cells (PBMCs) were conducted to characterize HuM291 and to establish an animal model. A multidose study was conducted in chimpanzees to evaluate the safety, pharmacokinetics, immunomodulatory activity, and immunogenicity of HuM291, when administered at doses ranging from 0.1 to 10 mg. RESULTS: HuM291 bound to and effectively downmodulated CD3 from chimpanzee PBMCs and stimulated substantially less cytokine secretion and proliferation of chimpanzee PBMCs compared with OKT3 (Orthoclone OKT3; Ortho Pharmaceutical Corp., Raritan, NJ). Multiple doses of HuM291 (0.1, 1.0, or 10 mg/dose) were not associated with adverse events, signs of toxicity, or CRS, despite cytokine release. HuM291 exhibited a long elimination t1/2 (81.5 hr) and, after three 10-mg doses, sustained serum concentrations > 1000 ng/ml were maintained for 1 week. Multiple 10-mg doses induced complete depletion of circulating CD2+CD3+ T cells for up to 10 days after the last dose; T cells recovered by Day 28. Anti-HuM291 Abs were observed in only 4 of 12 animals and were transient in 2 of those animals. CONCLUSIONS: In vitro, HuM291 is substantially less mitogenic than OKT3. In chimpanzees, HuM291 effectively depleted peripheral T cells without eliciting clinical signs of CRS, and recovered T cells were functionally normal.

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.

Pharmacology and safety assessment of humanized monoclonal antibodies for therapeutic use.

The humanization of monoclonal antibodies has generated a class of therapeutic products with improved safety, longer half-lives, and greatly diminished immunogenicity. These engineered proteins are highly species specific and in many cases only cross-react in humans. Where there is cross-reactivity in nonhuman primates or other species, it is not always clear that the pharmacologic effects reflect the potential actions in human volunteers or patients. As with other biologic products, the profile of humanized monoclonal antibodies dictates the preclinical strategy. The preclinical programs for the 2 humanized monoclonal antibodies described here, anti-HLA-DR (Hu1D10) and anti-CD3 (HuM291), demonstrate several unique aspects that affected their preclinical development strategy. Hu1D10 binds to a posttranslational form of HLA-DR and recognizes this antigen in some but not all human and nonhuman primates. The second antibody, HuM291, cross-reacts with CD3 only in the chimpanzee, which is not an optimal test species. In addition, a marketed anti-CD3 product exists (OKT3), and in the preclinical development of our antibody during testing of efficacy and safety, we needed to focus on adverse effects that might be similar to those of OKT3. In these studies, the safety, pharmacokinetics, immunogenicity, and pharmacology (B- and T-cell depletion and recovery) of the 2 antibodies were evaluated. The focus in this review is on the safety and pharmacology testing and the current status of each drug.

A study of complexes of class II invariant chain peptide: major histocompatibility complex class II molecules using a new complex-specific monoclonal antibody.

Complexes of major histocompatibility complex (MHC) class II molecules containing invariant chain (Ii)-derived peptides, known as class II-associated invariant chain peptides (CLIP), are expressed at high levels in presentation-deficient mutant cells. Expression of these complexes in mutant and wild-type antigen-presenting cells suggests that they represent an essential intermediate in the MHC class II antigen-presenting pathway. We have generated a monoclonal antibody, 30-2, which is specific for these complexes. Using this antibody, we have found quantitative differences in CLIP:MHC class II surface expression in mutant and wild-type cells. Our experiments also show that CLIP:MHC class II complexes are preferentially expressed on the cell surface similar to total mature MHC class II molecules. These complexes are found to accumulate in the endosomal compartment in the process of endosomal Ii degradation. Analysis of the fine specificity of the antibody indicates that these complexes have Li peptide bound to the peptide-binding groove.

A unique pattern of lymphokine synthesis is a characteristic of certain antigen-specific suppressor T cell clones.

We report that the lymphokines (IFN-gamma) and IL-10 are co-synthesized by previously described CD3+ TCR alpha beta+, minor antigen-specific suppressor T cell clones. IFN-gamma and IL-10 are known to (i) be characteristically produced by different helper T cell types, Th1 and Th2 respectively, and (ii) inhibit the function of the reciprocal subset of T cells: IFN-gamma inhibits the function of Th2 and IL-10 that of Th1 cells. Although Th0 cells are also known to synthesize cytokines of both the Th1- and Th2-type T cells, the suppressor T cells described in this report are different from Th0 cells in that they produce (i) neither IL-2 nor IL-4 molecules and (ii) stimulation via their CD3-TCR system seems independent of both IL-2 and IL-4, the typical autocrine molecules for T cell proliferation. The lymphokine profile of these suppressor T (Ts) cell clones, as well as those of human antigen-specific Ts cells reported earlier, suggests that co-synthesis of some Th1-like and some Th2-like cytokines may be a characteristic of antigen-specific Ts cells as opposed to the type of reciprocal inhibition mediated through IFN-gamma or IL-10, which is antigen non-specific.

A receptor for interleukin 10 is related to interferon receptors.

We isolated cDNAs encoding a mouse interleukin 10 receptor (mIL-10R) from mouse mast cell and macrophage cell lines. The two cDNAs are substantially identical and express an approximately 110-kDa polypeptide in COS7 cells, which binds mIL-10 specifically. A mouse pro-B-cell line (Ba/F3) expressing transfected recombinant mIL-10R binds IL-10 with high affinity (approximately 70 pM) and proliferates in response to mIL-10. mIL-10R is structurally related to interferon receptors (IFNRs). Since IL-10 inhibits macrophage activation by IFN-gamma, a possible implication of this relationship interaction of IL-10R and IFN-gamma R or their signaling pathways.

Dyspnea in dying patients.

Dyspnea is common in terminally ill patients and is often fairly difficult to control. If specific causes cannot be identified or treated, general measures to relieve symptoms should be used. Nondrug measures (eg, discussion and explanation with the patient) and drug measures (eg, morphine) can be used to control the dyspnea, although side effects, such as sedation, can be problematic.

Differential effects of IL-4 and IL-10 on IL-2-induced IFN-gamma synthesis and lymphokine-activated killer activity.

Culture of human peripheral blood mononuclear cells (PBMC) with IL-2 stimulates synthesis of cytokines and generation of lymphokine-activated killer (LAK) activity. Both IL-4 and IL-10 [cytokine synthesis inhibitory factor (CSIF)] inhibit IL-2-induced synthesis of IFN-gamma and tumor necrosis factor (TNF)-alpha by human PBMC. However, unlike IL-4, IL-10 inhibits neither IL-2-induced proliferation of PBMC and fresh natural killer (NK) cells, nor IL-2-induced LAK activity. Moreover, IL-4 inhibits IL-2-induced IFN-gamma synthesis by purified fresh NK cells, while in contrast the inhibitory effect of IL-10 is mediated by CD14+ cells (monocytes/macrophages). IL-10 inhibits TNF-alpha synthesis by monocytes or monocytes plus NK cells, but not by NK cells alone. These results suggest that IL-4 and IL-10 act on NK cells via distinct pathways, and that IL-2-induced cytokine synthesis and LAK activity are regulated via different mechanisms.

Interleukin 10 is a potent growth and differentiation factor for activated human B lymphocytes.

Interleukin 10 (IL-10), originally identified as a TH2 helper T-cell product able to inhibit cytokine production by TH1 cells, is highly homologous to BCRF1 (viral IL-10), an open reading frame in the Epstein-Barr virus genome. Here, we show that human and viral IL-10 stimulate DNA replication of B lymphocytes activated either via their antigen receptor or via their CD40 antigen. IL-4 and IL-10 display additive effects and induce a strong increase in the number of viable cells. Moreover, IL-10 induces activated B cells to secrete large amounts of IgG, IgA, and IgM, and the combination of IL-10 and IL-4 results in the secretion of the four immunoglobulin isotypes. Thus, IL-10 may play an important role in the amplification of humoral responses.

Expression of interleukin-10 activity by Epstein-Barr virus protein BCRF1.

Cytokine synthesis inhibitory factor (CSIF; interleukin-10), a product of mouse TH2 T cell clones that inhibits synthesis of cytokines by mouse TH1 T cell clones, exhibits extensive sequence similarity to an uncharacterized open reading frame in the Epstein-Barr virus BCRF1. Recombinant BCRF1 protein mimics the activity of interleukin-10, suggesting that BCRF1 may have a role in the interaction of the virus with the host's immune system.

Internal connectivity is pervasive among primary and secondary anti-hen egg white lysozyme (HEL) IgG monoclonal antibodies.

Twenty-one primary and 18 secondary monoclonal antibodies (mAb) with specificity for hen egg white lysozyme (HEL) were tested in pair-wise combinations for connectivity, that is, reactivity with each other in the absence of HEL, by an ELISA assay. All mAb reacted with at least one other, and a large proportion of the mAb, 7 of 16 primary and 11 of 18 secondary mAb, were reactive with 5 or more of the other mAb. Moreover, each gave a unique pattern of reactivity. Reactivity between mAb derived from primary and secondary hybridomas was also extensive. Some 'highly connective' mAb reacted with almost every other mAb tested. Two 'mini-networks' in the primary and secondary mAb, respectively, were identified through an ELISA binding assay and an inhibition study. Within these mini-networks (nets), members that bear IdC1 do not react with each other but bind to other members bearing IdC2. This type of interaction, as well as that between different nets defines a complicated network. No obvious correlation with fine specificity, presence of IdXE (a regulatory idiotype in the primary anti-HEL response), nor variable gene coding was apparent. Exact repeats of sequence were found for two pairs of mAb from independently selected hybridomas, i.e. in each case a primary and secondary hybridoma, indicating that high connectivity is associated with strong selection during B cell maturation following antigen stimulation.

A predominant idiotype independent of specificity, or Ig and H-2 allotypes, is found in the primary but not the secondary murine antibody response to lysozyme.

Removal of just the three N-terminal residues Lys-Val-Phe (TIP) on hen egg white lysozyme (HEL), by aminopeptidase cleavage, eliminates an antigenic determinant which is a recurrent and dominant focus of primary but not secondary antibody responses to HEL in a variety of mouse strains. We have generated an anti-idiotypic rabbit antiserum against such a TIP-dependent monoclonal antibody (mAb). This antiserum reacts with many different primary anti-HEL mAb, but fails to react with all of a variety of secondary anti-HEL mAb. The idiotype defined by this antiserum, termed IdXE, is a common feature of early anti-HEL antibody responses but does not appear in secondary responses. Although the presence of IdXE and TIP dependence is correlated in primary responses, studies of idiotype expression on mAb and on plaque-forming cells (PFC) using mixed erythrocyte monolayers clearly show that at the single-cell level the properties are separable, i.e., not all TIP-recognizing PFC display IdXE and a sizable proportion of cells producing non-TIP-dependent antibodies are IdXE+. The restricted idiotypy and specificity of early antibody responses to HEL occur in each of eight diverse mouse strains tested: it is not associated with a particular MHC haplotype, heavy chain allotype or light chain allotype. The finding of such strain-independent restriction in the early response pattern to a typical protein antigen is novel and suggests the involvement of highly conserved, potent regulatory mechanisms which are manifested as a limitation in the initial expression of the available repertoire.

Circular fringe carrier technique in holographic interferometry.

An improved fringe carrier technique can eliminate the ambiguity in holographic fringe interpretation. This paper presents a new fringe carrier technique-a circular fringe carrier technique that not only eliminates the ambiguity in holographic fringe interpretation but also determines the direction of displacements and obtains the required fringe spacings without precisely adjusting the object beam.