Idiotype switching of CD4-specific monoclonal antibodies can prolong the therapeutic effectiveness in spite of host anti-mouse IgG antibodies

Immunotherapy using murine monoclonal antibodies (mAb) is limited by the host anti-mouse IgG response. Previous investigations demonstrated that a large proportion of the anti-mouse response was specific for idiotypic determinants of the mAb. This study demonstrates the feasibility of idiotype switching of therapeutic mAb to evade this anti-idiotype response. In this way prolongation of the therapeutic effectiveness of mAb treatment can be achieved. Using different CD4-specific mAb consecutively in rhesus monkeys it was possible to obtain therapeutic effectiveness in spite of host anti-mouse IgG antibodies, provided that no anti-idiotypic antibodies were present. Anti-constant part antibodies may even enhance therapeutic effectiveness.     

Generation of anti-Neu-glycolyl-ganglioside antibodies by immunization with an anti-idiotype monoclonal antibody: A self versus non-self-matter

We have previously generated a murine anti-idiotype (Ab2) monoclonal antibody (mAb) to a murine Ab1 mAb, named P3, which selectively binds Neu-glycolyl (NeuGc)-sialic acid on several monosialo- and disialogangliosides, and also reacts with sulfatides and antigens expressed in human melanoma and breast tumors. This Ab2 mAb, designated as 1E10, induced anti-anti-idiotype antibodies (Ab3) in mice and cancer patients. These Ab3 generated by 1E10 mAb were characterized by bearing P3 mAb idiotopes (Ab3, Id +). But when the specificity of these Ab3 antibodies was tested, no specific humoral response against NeuGc-containing gangliosides was detected in sera from immunized mice. However, hyperimmune sera from melanoma and breast cancer patients vaccinated with this Ab2 mAb were able to react specifically with these gangliosides. The different expression of NeuGc-containing gangliosides in the normal tissues of mice and humans could explain these results. In order to demonstrate these findings in other animal species with a different NeuGc-sialic acid expression, we performed similar studies in monkeys and chickens. In monkeys, as in most mammals, NeuGc-containing gangliosides are self-antigens. In contrast, chickens, like humans, lack the expression of these antigens in normal tissues. Here we report that the antibody response against NeuGc-containing gangliosides induced by immunization with 1E10 mAb was completely different in both species. No specific antibody response against these gangliosides was detected in hyperimmune monkey sera. In contrast, a strong and specific Ab3 response against GM3(NeuGc) and GM2(NeuGc) gangliosides (Ab3, Ag+) was generated in chickens due to the administration of 1E10 mAb.     

Production of polyclonal and monoclonal antibodies against CD54 molecules by intrasplenic immunization of plasmid DNA encoding CD54 protein

DNA immunization, in theory, is of great interest as a source of specific antibodies against different antigens. In an attempt to produce polyclonal and monoclonal antibodies against cell surface molecules by using the DNA immunization strategy, intramuscular and intrasplenic routes of DNA injection were compared. Two to five, but not a single, intramuscular DNA immunizations induced anti-CD54 and anti-CD147 antibody production. In contrast, a single intrasplenic immunization of CD54-encoding DNA could induce anti-CD54 antibody production. To produce monoclonal antibody (mAb), spleen cells obtained from an intrasplenic CD54-encoding DNA immunized mouse were fused with myeloma cells using the standard hybridoma technique. A hybridoma secreting specific mAb to CD54 was established. The generated mAb reacted to CD54 protein expressed on transfected COS cells and various cell types, the same as using standard CD54 mAb MEM-111. Our results demonstrated that direct immunization of antigen-encoding DNA into spleen is an effective route for production of both polyclonal and monoclonal antibodies to cell surface molecules. This finding is very useful for the production of antibodies to cell surface molecules where the protein antigen is not available or difficult to prepare, but cDNA encoding the corresponding protein is available.     

Rhesus monkey responses to multiple injections of human monoclonal antibodies

Five rhesus monkeys were injected multiple times over several months with two different human IgG1 monoclonal antibodies, both directed against human cytomegalovirus. Three monkeys each injected four times with monoclonal antibody EV2-7 for over 200 days showed no response other than a normal decay in antibody level. The in vivo half life of this antibody was substantially longer when measured with an idiotype-specific two site immunoassay than with radiolabeled antibody, indicating that the iodination procedure greatly affected the stability of the antibody. Although there was considerable individual variation in the half-life of EV2-7, from 8.9 to 30.5 days, the half-life was fairly long, especially considering the size of the monkeys. Two monkeys were injected with monoclonal antibody EV1-15. One monkey has responded in a similar manner to the EV2-7-injected monkeys. However, the other monkey has produced an anti-idiotypic antibody (or antibodies) of high affinity. It is possible that this response was triggered by the unusual physical nature of antibody EV1-15 or the effect of the species difference between human and rhesus monkey. In any case, the results from these five monkeys indicate that human monoclonal antibodies should have a significant advantage over mouse monoclonal antibodies for in vivo therapeutic applications.     

Human and murine antibodies to rye grass pollen allergen LolpIV share a common idiotope.

The possibility that a murine monoclonal antibody (mAb 12) to Rye grass pollen allergen LolpIV and LolpIV-specific antibodies in the sera of grass allergic individuals share a common idiotope (Id) was investigated. It was first established that mAb 12 and human IgE antibodies recognized the same (or similar) epitope(s) on LolpIV; i.e. mAb 12 could inhibit, to the extent of 35-60%, the binding of 125I-LolpIV to the human IgE antibodies present in the sera of grass pollen-allergic individuals. Subsequently, a rabbit anti-Id antiserum was produced against mAb 12 and rendered Id-specific by appropriate immune absorptions, and its IgG antibody fraction was isolated (Rb-aId). The specificity of Rb-aId was demonstrated by the fact that the antibodies bound only to mAb 12 and not to any other murine monoclonal antibody tested. Observations that Rb-aId inhibited the binding of 125I-LolpIV to mAb 12 indicated that the Id determinants recognized on mAb 12 were located at or near the antibody-combining sites. The Rb-aId also bound specifically to affinity-purified human anti-LolpIV antibodies isolated from human sera, but not to affinity-purified human anti-tetanus toxoid antibodies. This indicated that the human anti-LolpIV antibodies share a cross-reactive Id. The binding of Rb-aId to human anti-LolpIV antibody could also be inhibited by mAb 12. Therefore, it was concluded that the murine and human antibodies to LolpIV share a cross-reactive idiotope.     

Differentiation antigens on rhesus monkey lymphocytes. I. Identification of T cells bearing CD3 and CD8, and of a subset of CD8-bearing cells

Rhesus monkeys provide an excellent preclinical model to test the effect of monoclonal antibodies (mAb) in vitro and in vivo. So far, mostly mAb have been used which were originally raised against human cell surface antigens but cross-reacted reasonably well with homologous antigens on rhesus monkey cells. However, to optimize the model, it was necessary to produce mAb which react specifically with subsets of rhesus monkey lymphocytes. In this report, three mouse anti-rhesus monkey mAb are described, specific for different subsets of rhesus monkey T lymphocytes. None of the reagents cross-reacts with human lymphocytes. Characterization of these mAb was based upon indirect immunofluorescence, using a simultaneous staining technique, and immunoprecipitation of the specific target antigens. One antibody (GM9) reacts with the same subset as is recognized by mAb specific for human CD8+ cells. The second mAb (GM13) is specific for a subset of CD8+ cells. A third mAb (FN18) was of particular interest: it identifies a cell surface complex, RhT3, expressed on mature T lymphocytes, of which the polypeptide chains have a molecular mass of 22 and 27 kDa. The data strongly suggest that RhT3 is a CD3-like determinant, so far unidentified in the rhesus monkey.     

Production and characterization of two murine monoclonal antibodies directed against epitopes exclusive to soluble fragments of FcεRII/CD23

Two murine monoclonal antibodies (mAb) designated as SU1 and SU3 directed against soluble FcεRII/CD23 have been generated by fusing X.63.AG.8653 (a mouse myeloma cell line) with spleen cells from mice immunized with an Epstein Barr virus (EBV)-transformed B cell line (RPMI-8866). The antibodies have been shown to be capable of detecting affinity purified soluble FcεRII/CD23 in an enzyme-linked immunosorbent assay. Indirect immunofluorescence has shown that the SU1 and SU3 mAb do not stain RPMI-8866, a FcεRII/CD23⁺ B cell line. By studying the migration profiles of affinity purified SU1- and SU3-reactive molecules on sodium dodecylsulfate-polyacrylamide gel electrophoresis it has been shown that SU1 mAb immunoprecipitates 33- and 12-kDa components, while the SU3 mAb recognized 25- and 45-kDa proteins from culture supernatants of RPMI-8866 cells. Moreover, affinity purified SU1- and SU3-reactive proteins have been shown to be recognized by human IgE but not by the human IgG molecule. These results provide evidence that SU1 and SU3 mAb may recognize some putative post-cleavage epitopes on the N-terminal end of the low affinity receptor which appear, perhaps, following the process of fragmentation. In addition, the effect of these antibodies on continuous growth of a panel of lymphoblastoid cell lines indicates that SU1 mAb was found incapable of influencing the spontaneous proliferation of EBV-immortalized B cell lines; whereas SU3 mAb completely blocked the spontaneous growth and proliferation of all B cell lines tested. The results are discussed in relation to the appearance of a functional post-cleavage epitope on soluble FcεRII/CD23.     

Enhancement of neutralizing efficacy by combining three monoclonal antibodies to human interferon-alpha.

Three murine monoclonal antibodies (mAb) directed to distinct epitopes on recombinant human interferon (IFN)-alpha 1, and three mAb recognizing distinct epitopes on recombinant human interferon (IFN) alpha 1, and three mAb recognizing distinct epitopes on recombinant human IFN-alpha sc, were studied by IFN-neutralizing assays. The efficacy of neutralization of the anti-viral and the anti-proliferative activities of IFN-alpha 1, or IFN-alpha 2c, by the specific antibodies used, individually or in combination, were evaluated. In comparison with single mAb, the mixtures of three mAb against IFN-alpha 1 or three mAb against IFN-alpha 2c were capable of neutralizing more than 10-times larger amounts of IFN-alpha 1 and alpha 2c, respectively. The strong potentiation of the neutralization efficacy resulting from mixing different mAb was demonstrated by neutralization of the anti-viral as well as the anti-proliferative activities of both recombinant IFN. The neutralization experiments support the interpretation that the observed potentiation results from simultaneous interaction of anti-IFN mAb with different epitope specificity.     

Studies on the monocyte dependence of T-cell proliferation induced by monoclonal antibodies directed against regions I and II of the CD2 antigen.

We produced three murine monoclonal antibodies (mAb) directed against the human T-cell differentiation antigen CD2 (formerly T11) and analysed their epitope specificity by E-rosette inhibition, cross-blocking and proliferation-induction experiments. When added together, two mAb (both IgG1 kappa) were able to induce proliferation in peripheral blood T cells. This proliferation was found to be strictly dependent on the presence of monocytes. The polymorphism of Fc receptors (FcR) expressed on human monocytes for murine IgG1 antibodies, which is readily demonstrable in anti-CD3-induced T-cell activation, was not found in the anti-CD2-driven system. Moreover, mAb directed against the 40,000 MW FcRII were unable to inhibit proliferation induced by the mitogenic anti-CD2 combination. Still, F(ab')2 fragments of the mitogenic anti-CD2 antibody combination could not initiate T-cell mitogenesis, despite their ability to induce a rise in the free intracellular [Ca2+]. The contributions of monocytes and of antibody Fc moieties to T-cell proliferation, induced by combinations of anti-CD2 mAb, will be discussed.     

Properties and pharmacokinetics of two humanized antibodies specific for L-selectin.

BACKGROUND: The participation of L-selectin in leukocyte recruitment during inflammation has suggested the use of L-selectin inhibitors as potential anti-inflammatory therapeutics. Blocking monoclonal antibodies could serve as such therapeutic agents, particularly if humanized to reduce their immunogenicity and improve their serum half-life. OBJECTIVES: For this purpose, two mouse monoclonal antibodies, DREG-55 and DREG-200, that block human L-selectin were humanized and characterized. STUDY DESIGN: The resulting humanized antibodies, HuDREG-55 and HuDREG-200, constructed with human IgG4 constant regions, were evaluated for their specificity, affinity and ability to block L-selectin-dependent adhesion in in vitro assays. Their pharmacokinetic behavior in rhesus monkeys was also studied. RESULTS: HuDREG-55 and HuDREG-200 were found to retain the specificity and affinity, within 2-fold, of the parent murine antibodies. HuDREG-55 and HuDREG-200 block L-selectin-dependent adhesion of human lymphocytes to high endothelial venules in frozen sections of lymph nodes. In addition, HuDREG-55 and HuDREG-200 are inhibitory in a novel L-selectin-dependent adhesion assay. This assay utilizes flow cytometry to measure binding of polymerized liposomes containing an analog of sialyl Lewis X, sialyl Lewis X glycoliposomes, to peripheral blood neutrophils and lymphocytes. Studying the pharmacokinetics of HuDREG-55 and HuDREG-200 in rhesus monkeys showed terminal elimination half-lives at 12.0 and 20.3 days, respectively. CONCLUSION: The shorter terminal elimination half-life of HuDREG-55 in rhesus monkeys may be due to the ability of HuDREG-55 but not HuDREG-200 to bind rhesus monkey L-selectin.     

Monoclonal antibodies that identify the CD3 molecules expressed specifically at the surface of porcine gammadelta-T cells

The CD3 antigen is a surface structure associated with the T-cell receptor (TCR) to form a complex involved in antigen recognition and signal transduction. Reports on the structures of the CD3 molecules associated with alphabeta- and gammadelta-TCR have been contradictory. To investigate this issue, we raised a panel of monoclonal antibodies (mAb) against purified porcine CD3 molecules. Unlike the conventional anti-CD3, these mAb reacted specifically with peripheral gammadelta-T cells, but not with alphabeta-T cells. Immunoprecipitation showed that the antibody recognized a subset of CD3 molecules that were associated with gammadelta-TCR. Also unlike the conventional anti-CD3, these mAb, though directed at two different epitope groups, failed to induce antigenic modulation, T-cell proliferation and CD3-redirected cytotoxicity. Taken together, these results suggest that there are differences in the antigenicity, signal transduction potentials and probably structural differences between the CD3 molecules expressed at the surface of alphabeta- and gammadelta-T cells.     

The expression of murine eutaneous late phase reaction requires both IgE antibodies and CD4 T eells

Background Exposure of atopic patients to a specific allergen evokes an immediate response which is followed, in many cases, by a late phase reaction (LPR) some hours later. Here we have examined the immunological mechanisms required for the expression of cutaneous LPR in mice. Methods BALB/c mice were immunized by i.p. injection of ovalbumin (OVA) and alum actively or by i.v. injection of anti-OVA IgE monoclonal antibody (mAb) passively. After challenge by intradermal injection of OVA into ears, the changes in ear thickness, the number of eosinophils, and the levels of IL4 and IFN-γ protein at the site of antigen challenge were examined. Results Actively immunized mice developed a biphasic response at the site of OVA injection, while mice passively immunized with IgE anti-OVA mAb displayed a strong early response but no LPR. Cell transfer experiments using BALB/c nu/nu mice revealed that both OVA-specific IgE mAb and OVA-primed CD4 T cells were required to evoke LPR. Moreover, LPR was associated with increased levels of IL-4 production concomitant with reduced IFN-γ production and was abolished by pretreatment with anti-IL-4 neutralizing mAb. Conclusion It is suggested that murine cutaneous LPR against OVA is a type 2 inflammatory response in which both IgH antibodies and CD4 T cells play an obligatory role.     

Activation of macaque T cells and B cells with agonistic monoclonal antibodies

A series of mouse monoclonal antibodies (mAb) to human differentiation antigens known to have agonistic activity for human T or B cells was found to bind specifically to macaque T or B cell subsets. Most of these mAb also stimulated macaque lymphocyte proliferation, implying that they recognize functional homologues in monkeys. Anti-CD3, anti-CD28 (9.3), and anti-Lp220 (CD45R) mAb stimulated proliferation of both human and macaque T cells; similarly, anti-IgM and anti-CDw40 mAb stimulated both human and macaque B cells. In contrast, anti-CD20 and anti-CD39 mAb, which are known to stimulate human B cells, did not stimulate macaque B cells. A human low-molecular weight B cell growth factor (BCGF) and anti-IgM were co-stimulatory for macaque splenic B cells but not for blood B cells, suggesting that B cell subpopulations may differ in their responsiveness to BCGF. The results show that functional epitopes on some lymphocyte surface molecules such as CD28 or CDw40 are conserved in primate evolution. Functional epitopes on other cell surface molecules such as CD3 and CD20 may have more complex evolutionary constraints.     

Allergen-specific polyclonal antibodies reduce allergic disease in a mouse model of allergic asthma

BACKGROUND: Recombinant allergen-specific immunoglobulin G (IgG) antibody therapy can reduce allergic asthma symptoms by inhibiting the immunoglobulin E (IgE)-mediated allergic response. This study investigated the effect of intranasally administered allergen-specific monoclonal (mAb) and polyclonal (pAb) antibody on airway inflammation and hyperresponsiveness (AHR) in a mouse model of human asthma. METHODS: Ovalbumin (OVA)-specific IgG2b antibodies were generated by phage display using spleens from OVA-immunized mice, and screening against OVA and finally expressed in CHO cells. Sensitized mice were treated intranasally with either a recombinant anti-OVA mAb (gc32) or a polyclonal preparation comprising seven selected antibodies (including gc32). Control mice received diluent only, OVA only, a control polymeric IgG or dexamethasone. Following challenge with nebulized OVA, investigators assessed airway inflammation by histology and cellular composition of the bronchoalveolar fluid, and methacholine-induced airway hyperresponsiveness (AHR). Serum levels of total and OVA-specific IgE were measured by ELISA. RESULTS: Sensitized mice developed airway inflammation and AHR in response to OVA challenge. Intranasally administered OVA-specific murine polyclonal or monoclonal IgG2b antibodies both reduced OVA-induced lung inflammation. Polyclonal, but not anti-OVA mAb, also reduced AHR and eosinophil influx into the airway lumen. Both anti-OVA antibody preparations reduced levels of specific IgE with no effect on total IgE levels. CONCLUSIONS: Intranasal treatment with allergen-specific pAb reduces pulmonary inflammation and AHR in a mouse model of allergic asthma, but allergen-specific mAb reduces inflammation only. Allergen-specific recombinant pAb offers a potentially valuable therapeutic approach to the management of allergic asthma.     

Immunomodulating antibodies in the treatment of metastatic melanoma: The experience with anti-CTLA-4, anti-CD137, and anti-PD1

Clinical activity of anti–CTLA-4 (cytotoxic T-lymphocyte antigen-4) monoclonal antibodies (mAb) has changed the approaches for the treatment of cancer in terms of patterns of response, duration of response, and adverse event profiles. In fact, antibodies that block the interaction of CTLA-4 with its ligands B7.1 and B7.2 can enhance immune responses, including anti-tumor immunity. Two recent studies using ipilimumab (an anti-CTLA-4 mAb) demonstrated improvements in overall survival in the treatment of advanced melanoma. These studies utilized two different schedules of treatment in different patient categories (first and second line of treatment). However, the results were quite similar despite the different dosage used and the combination with dacarbazine in the first line treatment. Ongoing clinical studies will establish the efficacy of ipilimumab as monotherapy or in combination with other drugs for the treatment of metastatic melanoma and a variety of other cancers. Other antibodies, such as CD137 agonists and PD-1 antagonists, are currently in various stages of pre-clinical and clinical development. Agonist antibodies directed against CD137 (4–1BB) on the surface of antigen-primed T-lymphocytes increase tumor immunity that is curative against some transplantable murine tumors. Programmed death-1 (PD1) is a surface molecule delivering inhibitory signals important to maintain T-cell functional silence against their cognate antigens. Interference with PD1 or its ligand PD-L1 (B7–H1) increases anti-tumor immunity. As a result, human mAbs anti-PD1 and anti-PD-L1 are under clinical development. This paper reviews recent studies in the treatment of advanced melanoma with these types of monoclonal antibodies. Ipilimumab can be considered a cornerstone of a new era in melanoma treatment. However, the aim is to optimize the therapy with anti-CTLA-4 antibodies to define the best schedule for next combination regimens (other immunomodulatory antibodies, BRAF/MEK inhibitors, vaccines, etc.) that represent the natural evolution of future melanoma therapy.     

Inhibition of human interleukin 4-induced IgE synthesis by a subset of anti-CD23/Fc epsilon RII monoclonal antibodies.

Specific monoclonal antibodies (mAb) directed against the CD23 antigen were used to study human interleukin 4 (hIL4)-induced IgE production by blood and tonsillar mononuclear cells. Both peripheral blood and tonsillar mononuclear cells stimulated by hIL4 expressed membrane CD23 as detected by the binding of all anti-CD23 mAb. Nevertheless, two sets of anti-CD23 mAb could be distinguished. The first set, including mAb 25, was able to decrease significantly hIL4-induced IgE synthesis by mononuclear cells. The second set, including EBVCS#1, did not affect hIL4-induced IgE synthesis. All the anti-CD23 mAb were able to bind specifically to a human B cell line expressing recombinant CD23. Inhibition experiments revealed that the two sets of anti-CD23 mAb did not recognize the same epitope on the CD23 antigen. In fact, all the anti-CD23 mAb, except EBVCS#1, were able to inhibit IgE binding to CD23 on RPMI 8866 cells. Moreover, the first set of antibodies, which decreased IgE production, was able to up-regulate membrane CD23 expression on hIL4-stimulated tonsillar mononuclear cells. Conversely, EBVCS#1, which had no effect on IgE production, did not affect hIL4-induced CD23 expression. These results indicate that CD23 plays a key role in human IgE synthesis.     

Immunomodulating antibodies in the treatment of metastatic melanoma: The experience with anti-CTLA-4, anti-CD137, and anti-PD1

Clinical activity of anti-CTLA-4 (cytotoxic T-lymphocyte antigen-4) monoclonal antibodies (mAb) has changed the approaches for the treatment of cancer in terms of patterns of response, duration of response, and adverse event profiles. In fact, antibodies that block the interaction of CTLA-4 with its ligands B7.1 and B7.2 can enhance immune responses, including anti-tumor immunity. Two recent studies using ipilimumab (an anti-CTLA-4 mAb) demonstrated improvements in overall survival in the treatment of advanced melanoma. These studies utilized two different schedules of treatment in different patient categories (first and second line of treatment). However, the results were quite similar despite the different dosage used and the combination with dacarbazine in the first line treatment. Ongoing clinical studies will establish the efficacy of ipilimumab as monotherapy or in combination with other drugs for the treatment of metastatic melanoma and a variety of other cancers. Other antibodies, such as CD137 agonists and PD-1 antagonists, are currently in various stages of pre-clinical and clinical development. Agonist antibodies directed against CD137 (4-1BB) on the surface of antigen-primed T-lymphocytes increase tumor immunity that is curative against some transplantable murine tumors. Programmed death-1 (PD1) is a surface molecule delivering inhibitory signals important to maintain T-cell functional silence against their cognate antigens. Interference with PD1 or its ligand PD-L1 (B7-H1) increases anti-tumor immunity. As a result, human mAbs anti-PD1 and anti-PD-L1 are under clinical development. This paper reviews recent studies in the treatment of advanced melanoma with these types of monoclonal antibodies. Ipilimumab can be considered a cornerstone of a new era in melanoma treatment. However, the aim is to optimize the therapy with anti-CTLA-4 antibodies to define the best schedule for next combination regimens (other immunomodulatory antibodies, BRAF/MEK inhibitors, vaccines, etc.) that represent the natural evolution of future melanoma therapy.     

Characterization of two CD23 monoclonal antibodies with reactivity distinct from other antibodies within this cluster of differentiation.

We have produced two CD23 monoclonal antibodies (mAb), LA1 and LA2, which differ significantly in their patterns of reactivity compared to other mAb within this cluster. Unlike other CD23 mAb, LA1 and LA2 show virtually no reactivity with freshly isolated tonsil B lymphocytes or mantle zone lymphocytes in tissue section. That LA1 and LA2 are CD23 mAb is confirmed by their precipitation of a 45,000 MW surface protein from B cells and strong reactivity with a CD23 transfectant. Cross-blocking studies with four well-characterized CD23 mAb show that LA1 and LA2 recognize the same, distinct epitope of the CD23 molecule. However, similar to other CD23 mAb, expression of LA1 and LA2 increases after activation. Following removal of cells staining with the well-characterized CD23 mAb MHM6, using a highly efficient magnetic bead technique, LA1 and LA2, but not other CD23 mAb, react with a subpopulation of the remaining cells when activated with interleukin-4 (IL-4) or phorbol ester. Soluble LA1 and MHM6 both provide a co-stimulatory signal for phorbol ester-induced B-cell proliferation. This response is increased if these mAb are used to cross-link the CD23 molecule. Interestingly, despite the fact that LA2 and LA1 cross-block, LA2 has no effect on functional responses in its soluble form but can elicit a comparable increase in proliferation when cross-linked. Results presented here suggest that the novel CD23 mAb, LA1 and LA2, recognize a distinct form of the CD23 molecule, expressed only on activation. These mAb define a subpopulation of activated B cells which do not stain with other CD23 mAb.     

CD23 and CD21 function as adhesion molecules in homotypic aggregation of human B lymphocytes

We have previously found that interleukin-4 and CD40 monoclonal antibodies (mAb) are strong potentiatiors of homotypic B cell aggregation which is dependent on LFA-1. We show here that CD23 mAb were also able to inhibit aggregation to a similar extent as LFA-1 antibodies. This inhibition was restricted to the MHM6 epitope of CD23 and antibodies to other epitopes [Epstein-Barr virus (EBV) CS-1, EBV CS-2, EBV CS-5 and mAb 25] or occupation of the Fc-binding site by IgE had no or a slightly enhancing effect on aggregation. When testing two antibodies to CD21, the recently defined ligand for CD23, one of these (BU32) was found to be inhibitory whereas the other (THB5) had no effect. By combining antibodies to LFA-1 and CD23, aggregation was often completely inhibited. These data suggest that LFA-1/ICAM-1 and CD23/CD21 are the major molecules involved in homotypic aggregation of human B cells.     

Adhesion molecule monoclonal antibodies inhibit experimental autoimmune thyroiditis.

To examine the role played by adhesion molecules in thyroid autoimmunity, we have assessed the effect of administering monoclonal antibodies (mAb) against intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) in experimental autoimmune thyroiditis, induced by immunizing rats with thyroglobulin in complete Freund's adjuvant. The antibody against LFA-1, but not against ICAM-1, reduced thyroglobulin antibody production (P < 0.01) and both antibodies caused a significant reduction (P < 0.002) in the severity of the thyroidal lymphocytic infiltration. In vitro, both mAb impaired the proliferative response of splenic and lymph node T cells to thyroglobulin, but only the antibody against LFA-1 reduced thyroid cell killing assessed using splenic lymphocytes as effectors. Monoclonal antibodies against both these adhesion molecules appear to inhibit cell-mediated autoimmunity in vivo, but only the LFA-1 mAb reduced the autoantibody response.