Role of CD8(+) lymphocytes in control of simian immunodeficiency virus infection and resistance to rechallenge after transient early antiretroviral treatment.

Transient antiretroviral treatment with tenofovir, (R)-9-(2-phosphonylmethoxypropyl)adenine, begun shortly after inoculation of rhesus macaques with the highly pathogenic simian immunodeficiency virus (SIV) isolate SIVsmE660, facilitated the development of SIV-specific lymphoproliferative responses and sustained effective control of the infection following drug discontinuation. Animals that controlled plasma viremia following transient postinoculation treatment showed substantial resistance to subsequent intravenous rechallenge with homologous (SIVsmE660) and highly heterologous (SIVmac239) SIV isolates, up to more than 1 year later, despite the absence of measurable neutralizing antibody. In some instances, resistance to rechallenge was observed despite the absence of detectable SIV-specific binding antibody and in the face of SIV lymphoproliferative responses that were low or undetectable at the time of challenge. In vivo monoclonal antibody depletion experiments demonstrated a critical role for CD8(+) lymphocytes in the control of viral replication; plasma viremia rose by as much as five log units after depletion of CD8(+) cells and returned to predepletion levels (as low as <100 copy Eq/ml) as circulating CD8(+) cells were restored. The extent of host control of replication of highly pathogenic SIV strains and the level of resistance to heterologous rechallenge achieved following transient postinoculation treatment compared favorably to the results seen after SIVsmE660 and SIVmac239 challenge with many vaccine strategies. This impressive control of viral replication was observed despite comparatively modest measured immune responses, less than those often achieved with vaccination regimens. The results help establish the underlying feasibility of efforts to develop vaccines for the prevention of AIDS, although the exact nature of the protective host responses involved remains to be elucidated.

A nonhuman primate model for the selective elimination of CD8+ lymphocytes using a mouse-human chimeric monoclonal antibody.

Nonhuman primates provide valuable animal models for human diseases. However, studies assessing the role of cell-mediated immune responses have been difficult to perform in nonhuman primates. We have shown that CD8+ lymphocyte-mediated immunity in rhesus monkeys can be selectively eliminated using the mouse-human chimeric anti-CD8 monoclonal antibody cM-T807. In vitro, this antibody completely blocked antigen-specific expansion of cytotoxic T cells and decreased major histocompatibility complex class I-restricted, antigen-specific lysis of target cells but did not mediate complement-dependent cell lysis. In vivo administration of cM-T807 in rhesus monkeys resulted in near total depletion of CD8+ T cells from the blood and lymph nodes for up to 6 weeks. This depletion was not solely complement-dependent and persisted longer in adults than in juveniles. Preservation of B cell and CD4+ T cell function in monkeys depleted of CD8+ lymphocytes was demonstrated by their ability to develop humoral immune responses to the administered chimeric monoclonal antibody. Furthermore, during CD8+ lymphocyte depletion, monkeys developed delayed-type hypersensitivity reactions comprised only of CD4+ T cells but not CD8+ T cells. This CD8+ lymphocyte depletion model should prove useful in defining the role of cell-mediated immune responses in controlling infectious diseases in nonhuman primates.

Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes.

Clinical evidence suggests that cellular immunity is involved in controlling human immunodeficiency virus-1 (HIV-1) replication. An animal model of acquired immune deficiency syndrome (AIDS), the simian immunodeficiency virus (SIV)-infected rhesus monkey, was used to show that virus replication is not controlled in monkeys depleted of CD8+ lymphocytes during primary SIV infection. Eliminating CD8+ lymphocytes from monkeys during chronic SIV infection resulted in a rapid and marked increase in viremia that was again suppressed coincident with the reappearance of SIV-specific CD8+ T cells. These results confirm the importance of cell-mediated immunity in controlling HIV-1 infection and support the exploration of vaccination approaches for preventing infection that will elicit these immune responses.

Minimal tumor necrosis factor receptor binding protein: optimum biological activity of a truncated p55 soluble tumor necrosis factor receptor-IgG fusion protein.

We have previously demonstrated, using expressed deletion constructs, that the fourth membrane proximal cysteine-rich repeat of the p55 TNF receptor (TNF-R) is not required for binding of tumour necrosis factor-alpha (TNF) or lymphotoxin-alpha (LT; tumour necrosis factor-beta). We and others have also shown that the soluble p55 TNF-R, rendered dimeric by fusion to an IgG backbone is extremely effective at neutralizing the harmful effects of TNF overproduction, such as in toxic shock. Here we address the question of how the TNF binding properties of the truncated TNF-R comprising the three distal cysteine-rich repeats (delta4 TNF-R), when fused with an IgG backbone, compare with those of the full length soluble receptor. We constructed several versions of the soluble delta4 TNF-R, on a complete IgG heavy chain backbone and on an IgG lacking the CH1 (first constant region) domain. The constructs were expressed with an Ig or native TNF receptor leader sequence and altered or native N terminal sequence, to compare efficiency of expression. When compared with a full length, soluble receptor Ig fusion protein, the affinity of all for TNF was identical, as were their activities in in vitro binding and cytotoxicity assays. In vivo studies showed that the delta4 and wild type fusion proteins afforded equivalent protection against LPS-induced lethality. However, the delta4 proteins exhibited a significantly lower affinity for LT, and reduced activity in LT binding and cytotoxicity assays. We conclude that the truncated TNF receptor IgG fusion protein is as effective at neutralizing TNF activity as the full length soluble receptor fusion protein. Its lower affinity for LT may make it a more selective agent in blocking the action of TNF, while causing less interference with the action of LT. Also its smaller size may make it a more useful therapeutic agent as it may be less immunogenic than the full length receptor.

Neutralization of TNF by the antibody cA2 reveals differential regulation of adhesion molecule expression on TNF-activated endothelial cells.

Upregulation of adhesion proteins plays an important role in mediating inflammation. The induction of adhesive molecules has been well studied, but the reversibility of their expression has not been well characterized. A neutralizing anti-TNF monoclonal antibody (cA2) was used to study the down regulation of TNF-induced E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on cultured human umbilical vein endothelial cells (HUVECs). Addition of cA2 following TNF stimulation of HUVECs enhanced the rate of E-selectin and VCAM-1 down-regulation from the cell surface and also reduced steady state E-selectin and VCAM-1 mRNA levels. The cA2-mediated disappearance of E-selectin, but not VCAM-1 protein was microtubule and not microfilament dependent. Neutralization of TNF only slightly reduced ICAM-1 cell surface levels following initial TNF stimulation, suggesting a slower turnover of ICAM-1 compared to E-selectin and VCAM-1. Microtubule inhibition during TNF stimulation partially inhibited E-selectin, VCAM-1 and ICAM-1 mRNA upregulation. VCAM-1 and ICAM-1 cell surface expression were similarly partially inhibited, however, E-selectin levels were unaffected, presumably due to the dual, opposing effect of inhibiting protein expression and inhibiting internalization. Microfilament inhibition during protein induction specifically inhibited the maximal expression of VCAM-1 protein and mRNA, without affecting E-selectin or ICAM-1. These data support the notion that E-selectin, VCAM-1, and ICAM-1 expression are differentially regulated on HUVECs and suggest that TNF neutralizing therapies may be effective because of their ability to reduce the levels of pre-existing adhesion proteins.

Inhibition of tumor necrosis factor activity minimizes target organ damage in experimental autoimmune uveoretinitis despite quantitatively normal activated T cell traffic to the retina.

Recent studies demonstrated that administration of a p55-tumor necrosis factor (TNF) receptor IgG-fusion protein (TNFR-IgG) prevented the clinical onset of experimental autoimmune encephalomyelitis but did not alter the number or tissue distribution of autoantigen-specific CD4+ effector T cells which trafficked into the central nervous system. To determine whether specific target tissues of autoimmune damage remain intact after TNFR-IgG treatment despite the presence of inflammatory cells within the tissues, we examined rats with experimental autoimmune uveoretinitis (EAU), as in this model, the main target of autoreactive CD4+ T cells, the retinal rod outer segments (ROS), can be examined readily by light microscopy. As judged by direct ophthalmoscopy, the onset of inflammation in the anterior chamber of the eye in EAU following administration of TNFR-IgG was delayed by 6 days compared to untreated controls, but the magnitude of the response was only slightly less than controls. Histological examination of the retinae and direct assessment of retinal inflammation revealed a disproportionate sparing of ROS in the TNFR-IgG-treated animals despite a level of retinal inflammation not substantially less than controls in which ROS damage was marked. Analysis of retinal leukocytes by immunofluorescence microscopy and flow cytometry indicated that approximately equal numbers of CD4+ alpha beta TCR+ lymphocytes were present in treated and control retinae, more than 30% of CD4+ cells in both experimental groups expressed the CD25 or MRC OX40 activation markers and most cells, which would include the CD4+ T lymphocytes, were activated as evidenced by MHC class II expression. Fewer activated macrophages and granulocytes were present in the treated retinae, possibly reflecting the lower level of tissue damage and subsequent accumulation of these inflammatory cells. The results demonstrate directly that a tissue specifically targeted for autoimmune destruction can be protected despite the influx of fully activated CD4+ T cells.

The immunogenicity of the 7E3 murine monoclonal Fab antibody fragment variable region is dramatically reduced in humans by substitution of human for murine constant regions.

A murine monoclonal antibody (7E3) directed against the platelet glycoprotein IIb/IIIa was engineered to reduce immunogenicity by substituting human for murine constant regions. The chimeric antibody is functionally identical to the murine antibody in vitro. Results from clinical trials with 7E3 Fab antibody fragments, however, show that the 7E3 variable region, which elicits the vast majority of the immune response to murine 7E3 Fab, is rendered dramatically less immunogenic (incidence reduced from 17% to 1%) when the identical variable region is linked to human rather than murine constant regions. Neither murine nor human constant regions were highly immunogenic themselves. We conclude that the constant regions of the Fab fragments are critical in modulating the immune response elicited by the linked 7E3 variable region. Because naturally occurring anti-human Fab fragment antibodies are prevalent both in the normal human population and in the patient population studied here, murine 7E3 Fab and chimeric 7E3 Fab may be fundamentally different in their interactions with the human immune system. This difference may be related to the dramatic difference in immunogenicity observed between murine 7E3 Fab and chimeric 7E3 Fab.

Functional comparisons of different tumour necrosis factor receptor/IgG fusion proteins.

Nine different IgG fusion proteins and one non-fusion protein, all containing sequences from the extracellular domain of either of two human TNF receptors, were compared for their ability to bind and inhibit human TNF-alpha or TNF-beta. The fusion proteins differed with respect to TNF receptor type (p55 or p75 TNF receptor), receptor valency (one, two or four receptor domains per molecule), the presence or absence of a CH1 domain in the IgG constant region, and the proportion of the extracellular domain included in the construct. In vitro TNF binding assays and cytotoxicity assays indicated that, of the constructs that bound TNF, the greatest difference in affinity and neutralizing capability was between monovalent and bivalent receptor constructs. Differences were also noted between tetravalent and bivalent versions of p55 fusion proteins, as well as between a p75 fusion protein comprising the complete extracellular domain and one lacking the C-terminal 53 amino acids of the extracellular domain. p55 constructs that included only the second cysteine-rich domain (CRD) or only the second and third CRDs showed no TNF binding activity. The presence or absence of an IgG CH1 domain made no difference in the ability of fusion proteins to neutralize TNF-alpha or TNF-beta. Animal experiments comparing the tetravalent and bivalent p55 fusions and the effects of the CH1 domain did not show significant differences in their ability to protect mice from endotoxin-induced lethality, although the p55 fusion proteins appeared to be more protective than the p75 fusion proteins. Thus, this study has identified structural modifications to TNF receptor/IgG fusion proteins which have differing effects on their neutralizing ability towards TNF-alpha or TNF-beta.

Unimpaired autoreactive T-cell traffic within the central nervous system during tumor necrosis factor receptor-mediated inhibition of experimental autoimmune encephalomyelitis.

The critical role of tumor necrosis factor (TNF) as a mediator in autoimmune inflammatory processes is evident from in vivo studies with TNF-blocking agents. However, the mechanisms by which TNF, and possibly also its homologue lymphotoxin alpha, contributes to development of pathology in rheumatoid arthritis and Crohn disease and in animal models like experimental autoimmune encephalomyelitis is unclear. Possibilities include regulation of vascular adhesion molecules enabling leukocyte movement into tissues or direct cytokine-mediated effector functions such as mediation of tissue damage. Here we show that administration of a TNF receptor (55 kDa)-IgG fusion protein prevented clinical signs of actively induced experimental autoimmune encephalomyelitis. Significantly, the total number of CD4+ T lymphocytes isolated from the central nervous system of clinically healthy treated versus diseased control animals was comparable. By using a CD45 congenic model of passively transferred experimental autoimmune encephalomyelitis to enable tracking of myelin basic protein-specific effector T lymphocytes, prevention of clinical signs of disease was again demonstrated in treated animals but without quantitative or qualitative impediment to the movement of autoreactive T lymphocytes to and within the central nervous system. Thus, despite the uninterrupted movement of specific T lymphocytes into the target tissue, subsequent disease development was blocked. This provides compelling evidence for a direct effector role of TNF/lymphotoxin alpha in autoimmune tissue damage.

Lipopolysaccharide (LPS) neutralizing peptides reveal a lipid A binding site of LPS binding protein.

Endotoxic shock follows a cascade of events initiated by release of lipopolysaccharide during infection with Gram-negative organisms. Two overlapping 15-mer peptides were identified, corresponding to residues 91-108 of human lipopolysaccharide binding protein that specifically bound the lipid A moiety of lipopolysaccharide with high affinity. The peptides inhibited binding of lipopolysaccharide to lipopolysaccharide binding protein, inhibited the chromogenic Limulus amebocyte lysate reaction, and blocked release of tumor necrosis factor alpha following lipopolysaccharide challenge both in vitro and in vivo. These results suggest lipopolysaccharide binding protein residues 91-108 form at least part of the lipopolysaccharide binding site. Moreover, derivatives of lipopolysaccharide binding protein residues 91-108 might modulate lipopolysaccharide toxicity in the clinical setting.

Chimeric anti-TNF-alpha monoclonal antibody cA2 binds recombinant transmembrane TNF-alpha and activates immune effector functions.

Results of clinical trials have indicated that cA2, a neutralizing mouse/human IgG1 chimeric anti-human TNF-alpha monoclonal antibody, may have therapeutic benefit for rheumatoid arthritis patients. Arthritic joints contain, in addition to elevated levels of soluble TNF-alpha, high numbers of CD4+ T cells and macrophages, cells known to express transmembrane TNF-alpha upon activation. For that reason, we sought to determine if cA2 binds to transmembrane TNF-alpha and what effects such binding may have on TNF-alpha-expressing cells. A cell line expressing a cell-surface, mutant form of transmembrane TNF-alpha was prepared for these studies. Analysis of these TNF+ cells by flow cytometry, direct binding, and competitive binding assays showed that cA2 binds to the transmembrane form of TNF-alpha with high avidity. Binding of the IgG1 isotype of cA2, but not an IgG4 version of cA2, resulted in efficient killing of the TNF+ cells by both antibody-dependent cellular toxicity and complement-dependent cytotoxicity effector mechanisms. These findings indicate that, in addition to blocking soluble TNF-alpha activity, cA2 can bind to transmembrane TNF-alpha in vitro and suggest that cA2 binding may lead to lysis of TNF-alpha-expressing cells in vivo.

Successful therapy of collagen-induced arthritis with TNF receptor-IgG fusion protein and combination with anti-CD4.

We have previously shown that anti-tumour necrosis factor (TNF) monoclonal antibody (mAb) ameliorates established collagen-induced arthritis and that the efficacy of this form of treatment can be enhanced by concurrent anti-CD4 treatment. Here we assess the efficacy of a human p55 TNF receptor-IgG fusion protein (p55-sf2), given alone or with anti-CD4 mAb. TNF receptor-IgG fusion protein (100 micrograms) suppressed paw swelling and limb recruitment in established arthritis and reduced the incidence of erosions in the proximal interphalangeal joints from 92% to 50%, which was comparable to 41% erosions using anti-TNF mAb. Methylprednisolone acetate (4.2 mg/kg/week) reduced clinical signs of inflammation in a manner comparable to TNF blockade but had little effect on the incidence of erosions. Co-administration of anti-CD4 and TNF receptor-IgG led to an even greater therapeutic effect than TNF receptor-IgG alone, with the incidence of erosions being reduced from 100% to 17%. Serological analyses showed that the beneficial effects of anti-CD4 and TNF receptor-IgG could be partly explained by the ability of anti-CD4 to prevent a neutralizing antibody response. These results confirm the importance of TNF in destructive inflammatory arthritis and demonstrate the feasibility of therapeutically targeting TNF with a form of TNF receptor. Finally, the findings confirm the beneficial effects of TNF-targeted therapy coupled with anti-CD4 therapy.

The mouse/human chimeric monoclonal antibody cA2 neutralizes TNF in vitro and protects transgenic mice from cachexia and TNF lethality in vivo.

The pleiotropic cytokine tumour necrosis factor-alpha (TNF) is thought to play a central role in infectious, inflammatory and autoimmune diseases. Critical to the understanding and management of TNF-associated pathology is the development of highly specific agents capable of modifying TNF activity. We evaluated the ability of a high affinity mouse/human chimeric anti-TNF monoclonal antibody (cA2) to neutralize the in vitro and in vivo biological effects of TNF. cA2 inhibited TNF-induced mitogenesis and IL-6 secretion by human fibroblasts, TNF-priming of human neutrophils, and the stimulation of human umbilical vein endothelial cells by TNF as measured by the expression of E-selectin, ICAM-1 and procoagulant activity. cA2 also specifically blocked TNF-induced adherence of human neutrophils to an endothelial cell monolayer. Receptor binding studies suggested that neutralization resulted from cA2 blocking of TNF binding to both p55 and p75 TNF receptors on the cells. In vivo, repeated administration of cA2 to transgenic mice that constitutively express human TNF reversed the cachectic phenotype and prevented subsequent mortality. These results demonstrated that cA2 effectively neutralized a broad range of TNF biological activities both in vitro and in vivo.

TNF receptor fusion proteins are effective inhibitors of TNF-mediated cytotoxicity on human KYM-1D4 rhabdomyosarcoma cells.

KYM-1D4 cells are a subline derived from a human rhabdomyosarcoma which are highly sensitive to TNF-mediated cytotoxicity. They were selected for this study because they express human TNF-R and are therefore a more relevant target for comparing the potential therapeutic value of human TNF-inhibitory agents than the usual murine cell lines. Two recombinant soluble TNF-R-IgG fusion proteins, one containing p55 TNR-R, the other containing p75 TNF-R, and a recombinant monomeric soluble p55 TNF-R were all found to block the cytotoxicity generated by human TNF-alpha and LT as well as also murine TNF. The p55 TNF-R-IgG fusion protein (p55-sf2) was the most effective of the antagonists tested, requiring an equimolar, (based on a monomeric configuration of TNF-alpha) or a 3-fold higher (based on a trimeric configuration of TNF-alpha) molar concentration to inhibit the cytotoxicity mediated TNF-alpha by 50%. p55-sf2 was also as effective at inhibiting the cytotoxicity mediated by LT or murine TNF in the KYM-1D4 assay. In contrast, the monomeric soluble p55 TNF-R was the least effective inhibitor, requiring a > 4000-fold higher molar concentration than p55-sf2 to achieve a similar degree of protection. The fusion proteins, particularly p55-sf2, may be useful as human therapeutic agents, as at low concentrations they can prevent both TNF-alpha-mediated and LT-mediated effects on human cells. As TNF-R-IgG fusion proteins also block the action of murine TNF in vitro, they may also be useful in the investigation of murine models of human inflammatory disease.

Expression and characterization of cM-T413, a chimeric anti-CD4 antibody with in vitro immunosuppressive activity.

Anti-CD4 monoclonal antibodies (mAbs) have shown considerable promise in the treatment of rheumatoid arthritis, psoriasis, and allograft rejection and may have potential use in blocking HIV-1 infection. One such anti-CD4 mAb we have developed, chimeric M-T412 (or cM-T412), has been used in clinical trials to treat rheumatoid arthritis, generalized postular psoriasis, and other autoimmune diseases. Here we report the cloning and expression of a second chimeric anti-CD4 mAb using M-T413, a murine mAb that blocks HIV-1 infection of H9 cells. We cloned the immunoglobulin light and heavy chain variable regions of M-T413, combined them with the human kappa (light chain) or G1, G2, G3 and G4 (heavy chain) constant regions in human expression vectors, and expressed these chimeric mAbs in 653 cells. Like chimeric M-T412 IgG1, the chimeric M-T413 mAbs inhibit T-cell proliferation in the mixed lymphocyte response and thus can act to immunosuppress CD4+ T-cell response. In contrast to M-T412, however, the M-T413 chimeric mAbs have reduced activity in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay using human CD4+ target and effector cells. We conclude that the chimeric M-T413 mAbs have potential utility in treating autoimmune disease and may be useful as prophylactics in preventing HIV-1 infection.

Treatment of rheumatoid arthritis with chimeric monoclonal antibodies to tumor necrosis factor alpha.

OBJECTIVE: To evaluate the safety and efficacy of a chimeric monoclonal antibody to tumor necrosis factor alpha (TNF alpha) in the treatment of patients with rheumatoid arthritis (RA). METHODS: Twenty patients with active RA were treated with 20 mg/kg of anti-TNF alpha in an open phase I/II trial lasting 8 weeks. RESULTS: The treatment was well tolerated, with no serious adverse events. Significant improvements were seen in the Ritchie Articular Index, which fell from a median of 28 at study entry to a median of 6 by week 6 (P < 0.001), the swollen joint count, which fell from 18 to 5 (P < 0.001) over the same period, and in the other major clinical assessments. Serum C-reactive protein levels fell from a median of 39.5 mg/liter at study entry to 8 mg/liter at week 6 (P < 0.001), and significant decreases were also seen in serum amyloid A and interleukin-6 levels. CONCLUSION: Treatment with anti-TNF alpha was safe and well tolerated and resulted in significant clinical and laboratory improvements. These preliminary results support the hypothesis that TNF alpha is an important regulator in RA, and suggest that it may be a useful new therapeutic target in this disease.

Use of a chimeric monoclonal anti-CD4 antibody in patients with refractory rheumatoid arthritis.

OBJECTIVES: To evaluate the safety, immunogenicity, and biologic effects of chimeric monoclonal anti-CD4 (cM-T412) in patients with refractory rheumatoid arthritis (RA), and to obtain preliminary data on the clinical response to this treatment. METHODS: Twenty-five patients with active refractory RA were treated with incremental doses (10 to 700 mg) of cM-T412 in an open-label, escalating-dose phase I trial. RESULTS: Infusion with cM-T412 was followed by an immediate, rapid decline in CD4+ T cells. The level of circulating CD4+ T cells remained depressed in most patients even at 6 months posttreatment. Following antibody infusion, proliferative responses of peripheral blood lymphocytes to mitogens and antigens were determined; mitogen and antigen responses were decreased compared with pretreatment responses. Mitogen responses tended to return to baseline values more rapidly than did responses to antigen. Adverse events included fever (19 patients), which was associated with myalgias, malaise, and asymptomatic hypotension; these symptoms were self-limited and appeared to correlate with transient elevations in interleukin-6. No significant human antibody response to the cM-T412 variable region was detected; only 2 patients developed transiently low levels of antibodies reactive with cM-T412. Significant clinical improvement, defined as > or = 50% decrease in tender joint counts compared with baseline, was noted in 43% of patients at 5 weeks and 33% at 6 months following cM-T412 infusion. CONCLUSIONS: Treatment of refractory RA with cM-T412 appears to be safe and is associated with sustained decreases in circulating CD4+ T cell counts and depressed in vitro T cell responses. No significant human antichimeric antibody response was detected. Nonblinded assessment of clinical end points suggests that treatment with cM-T412 may have beneficial effects in these patients with refractory RA. A double-blind clinical trial is warranted to determine its clinical efficacy in treating RA.

Effects of isotype and Fc region on in vitro function of a mouse/human chimeric CD4 antibody.

Murine CD4 mAbs have shown potential for the treatment of allograft rejection and autoimmune disorders including rheumatoid arthritis. Clinical usefulness of the murine mAbs has been limited by immunogenicity and a short circulating half-life. Mouse/human chimeric antibodies have been constructed, composed of the variable region of M-T412 (a murine G2a mAb specific for the human CD4 molecule) and human G1 (cM-T412 G1) or G4 (cM-T412 G4) Fc regions. F(ab')2 and F(ab) fragments of the murine G2a and chimeric G1 mAbs were generated by enzymatic digestion. The chimeric mAbs and all fragments retained the avidity and specificity of the murine M-T412 and were evaluated in in vitro assays measuring Ig production by pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cells (PBMC), sIL-2R produced by phytohemagglutinin-stimulated PBMC, and proliferation in response to tetanus toxoid, CD3 mAb plus IL-2, and mixed lymphocyte response (MLR). When PBMC were stimulated with tetanus toxoid, 10 ng/ml of cM-T412 G1 inhibited proliferation by 90%, while neither the cM-T412 G4, M-T412 G2a, nor any mAb fragment produced > 65% inhibition, even at 1000-fold higher concentrations. A similar pattern of inhibition was observed in MLR assays. In contrast, the F(ab')2 fragment of the cM-T412 G1 was as effective as the whole antibody in inhibiting PWM-stimulated IgM synthesis and PBMC proliferation in response to stimulation by a CD3 mAb plus IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)

High-level expression and characterization of a mouse-human chimeric CD4 antibody with therapeutic potential.

The use of murine anti-CD4 monoclonal antibodies (MAbs) has shown considerable promise for the treatment of allograft rejection and rheumatoid arthritis. We have constructed mouse-human anti-CD4 antibodies with the goal of increasing their clinical potential by decreasing immunogenicity and improving effector functions. The chimeric antibodies were constructed by cloning the heavy and light chain variable regions of M-T412, a murine antibody raised against the human CD4 antigen, and joining them to the human G1, G4, or kappa constant regions in mammalian expression vectors. After transfection into mouse myeloma cells, stable cell lines were isolated that secrete up to 140 micrograms/ml chimeric antibody in static culture. The chimeric antibodies were equivalent to the murine antibody in their binding characteristics and relative affinities. However, the chimeric M-T412 MAbs have enhanced activity when compared to the murine G2a MAb in mediating antibody-dependent cell-mediated cytotoxicity using human CD4+ target and effector cells.

Stable expression of cloned human antibody genes in murine myeloma cells.

Human monoclonal antibodies (MAbs) offer potential advantages over murine MAbs for therapy because they are not likely to elicit immune responses and are expected to interact more efficiently with the human immune system to activate therapeutically useful functions. Traditional methods for obtaining human MAbs (i.e., immortalization of B cells by cell fusion or transformation) can result in low and unstable antibody secretion. Recently, methods have been devised for direct cloning of human variable region genes via polymerase chain reaction and phage combinatorial libraries. Both types of human MAb production can benefit from expression systems that support the stable, high-level antibody secretion required for therapeutic use. Using an existing human-derived hybridoma that secretes a human IgM antibody as a convenient source of antibody genes, we have demonstrated that cloned human antibody genes can be efficiently expressed in murine myeloma cells and that cell lines with properties suitable for large-scale economical production can be obtained. We were unable to detect any differences between the antibodies produced by the original hybridoma and the engineered cell line. In addition, we were able to express an IgG form of the antibody, showing that expression of a recombinant human antibody need not be limited to the original antibody class.