Differential immunogenicity of two peptides isolated by high molecular weight-melanoma-associated antigen-specific monoclonal antibodies with different affinities

Peptide mimics isolated from phage display peptide libraries by panning with self-tumor-associated Ag (TAA)-specific mAbs are being evaluated as immunogens to implement active specific immunotherapy. Although TAA-specific mAb are commonly used to isolate peptide mimics, no information is available regarding the Ab characteristics required to isolate immunogenic TAA peptide mimics. To address this question, we have used mAb 763.74 and mAb GH786, which recognize the same or spatially close antigenic determinant(s) of the human high m.w.-melanoma-associated Ag (HMW-MAA), although with different affinity. mAb 763.74 affinity is higher than that of mAb GH786. Panning of phage display peptide libraries with mAb 763.74 and mAb GH786 resulted in the isolation of peptides P763.74 and PGH786, respectively. When compared for their ability to induce HMW-MAA-specific immune responses in BALB/c mice, HMW-MAA-specific Ab titers were significantly higher in mice immunized with P763.74 than in those immunized with PGH786. The HMW-MAA-specific Ab titers were markedly increased by a booster with HMW-MAA-bearing melanoma cells, an effect that was significantly higher in mice primed with P763.74 than in those primed with PGH786. Lastly, P763.74, but not PGH786, induced a delayed-type hypersensitivity response to HMW-MAA-bearing melanoma cells. These findings suggest that affinity for TAA is a variable to take into account when selecting mAb to isolate peptide mimics from a phage display peptide library.     

Specificity of mimotope-induced anti-high molecular weight-melanoma associated antigen (HMW-MAA) antibodies does not ensure biological activity

Vaccines based on peptide mimics (mimotopes) of conformational tumor antigen epitopes have been investigated for a variety of human tumors including breast cancer, tumors expressing the carcinoembryonic antigen, B cell lymphoma, neuroblastoma, and melanoma. In our previous work, we designed a vaccine based on a mimotope of the high molecular weight-melanoma associated antigen (HMW-MAA) that elicited HMW-MAA-specific antibodies (Abs) with anti-tumor activity in vitro and in vivo. In this study, we aimed to identify mimotopes of additional distinct HMW-MAA epitopes, since they could be used to construct a polymimotope melanoma vaccine. For this purpose, random peptide phage libraries were screened with the anti-HMW-MAA monoclonal antibodies (mAbs) VT80.12 and VF1-TP43 yielding one peptide ligand for each mAb. Both peptides inhibited the binding of the corresponding mAb to the HMW-MAA. Furthermore, when coupled to the carrier protein keyhole limpet hemocyanin (KLH), both HMW-MAA mimotopes elicited peptide-specific Abs in rabbits or BALB/c mice, but only the mimotope isolated with the mAb VT80.12 elicited HMW-MAA-specific Abs and only in mice. However, the latter Abs had no detectable effect on HMW-MAA expressing human melanoma cells in vitro. These results describe limitations related to the phage display technique and emphasize the need to characterize the functional properties of the mAb utilized to isolate mimotopes of the corresponding epitopes.     

CD4-dependent potentiation of a high molecular weight-melanoma-associated antigen-specific CTL response elicited in HLA-A2/Kb transgenic mice

The human high m.w.-melanoma-associated Ag (HMW-MAA) is an attractive target for the immunotherapy of melanoma, due to its relatively high expression in a high percentage of melanoma lesions and its restricted distribution in normal tissues. Active immunization with HMW-MAA mimics has been previously shown to induce a HMW-MAA-specific, T cell-dependent Ab response associated with an apparent clinically beneficial effect in advanced melanoma patients. Although T cells play an important role in controlling tumor growth, only limited information is available to date about the induction of HMW-MAA-specific CTL. In this report, we show that immunization of HLA-A2/K(b) transgenic mice with HMW-MAA cDNA-transfected syngeneic dendritic cells elicited a CD8(+) CTL response specific for HMW-MAA peptides with HLA-A2 Ag-binding motifs. The elicited CTL lysed HLA-A2(+)HMW-MAA(+) melanoma cells in vitro, and mouse HLA-A2/K(b) cells pulsed with HMW-MAA-derived peptides in vitro and in vivo. Although this CTL response could be generated in the absence of CD4(+) T cell help, harnessing CD4(+) T cell help in a noncognate Ag-specific manner with the polyclonal activator staphylococcal enterotoxin A augmented the CTL response. These results imply that dendritic cell-based immunization, in combination with CD4(+) T cell help, represents an effective strategy to implement T cell-based immunotherapy targeting HMW-MAA in patients with HMW-MAA-bearing tumors.     

Vaccination with a human high molecular weight melanoma-associated antigen mimotope induces a humoral response inhibiting melanoma cell growth in vitro

Peptide mimics of a conformational epitope that is recognized by a mAb with antitumor activity are promising candidates for formulations of anticancer vaccines. These mimotope vaccines are able to induce a polyclonal Ab response focused to the determinant of the mAb. Such attempts at cancer immunotherapy are of special interest for malignant melanoma that is highly resistant to chemotherapy and radiotherapy. In this study, we describe for the first time the design and immunogenicity of a vaccine containing a mimotope of the human high m.w. melanoma-associated Ag (HMW-MAA) and the biological potential of the induced Abs. Mimotopes were selected from a pVIII-9mer phage display peptide library with the anti-HMW-MAA mAb 225.28S. The mimotope vaccine was then generated by coupling the most suitable candidate mimotope to tetanus toxoid as an immunogenic carrier. Immunization of rabbits with this vaccine induced a specific humoral immune response directed toward the epitope recognized by the mAb 225.28S on the native HMW-MAA. The induced Abs inhibited the in vitro growth of the melanoma cell line 518A2 up to 62%. In addition, the Abs mediated 26% lysis of 518A2 cells in Ab-dependent cellular cytotoxicity. Our results indicate a possible application of this mimotope vaccine as a novel immunotherapeutic agent for the treatment of malignant melanoma.     

Analysis of the interaction between a human high molecular weight melanoma-associated antigen and the monoclonal antibodies to three distinct antigenic determinants

The restricted tissue distribution and the limited heterogeneity that appear in melanoma lesions of the high M.W. melanoma-associated antigen (HMW-MAA) suggest that this antigen may be an appropriate marker for radioimaging, and a useful target for immunotherapy in patients with melanoma. Therefore, in this study we analyzed other characteristics that are important in the selection of reagents for radioimaging and immunotherapy purposes. The affinities of the monoclonal antibodies (MoAB) 149.53, 225.28S, and 763.74T to distinct determinants of the HMW-MAA were found to be at least 1 X 10(8) mol/L. Furthermore, the effects of the concentrations of unlabeled MoAb on the dissociation rates suggest that the binding of MoAb 149.53 and 225.28S to melanoma cells (Colo 38) is preferentially bivalent, whereas that of MoAb 763.74T is preferentially univalent. These results suggest that the latter MoAb is the reagent of choice for assays that make use of soluble HMW-MAA, whereas the former two are the reagents of choice for assays with membrane-bound HMW-MAA, such as imaging with radiolabeled MoAb. The density of the HMW-MAA on cultured Colo 38 melanoma cells appears to be in the range of approximately 5 X 10(6) molecules/cell. The HMW-MAA was not susceptible to MoAb-mediated modulation under a variety of experimental conditions that included various concentrations of modulating MoAb, different incubation times, the use of an anti-mouse Ig antiserum, and the relaxation of equilibrium by diluting cells in MoAb-free medium. These results indicate that the HMW-MAA and the available corresponding MoAb meet the criteria to be reagents for radioimaging and immunotherapy in patients with melanoma.     

Targeting 11q23 positive acute leukemia cells with high molecular weight-melanoma associated antigen-specific monoclonal antibodies

Background  Acute leukemia with 11q23 aberrations is associated with a poor outcome with therapy. The lack of efficacy of conventional therapy has stimulated interest in developing novel strategies. Recent studies have shown that 11q23-positive acute leukemia cells express the high molecular weight-melanoma associated antigen (HMW-MAA). This tumor antigen represents a useful target to control growth of human melanoma tumors in patients and in severe combined immunodeficient (SCID) mice, utilizing antibody-based immunotherapy. This effect appears to be mediated by inhibition of the HMW-MAA function such as triggering of the focal adhesion kinase/proline-rich tyrosine kinase 2 (Pyk2) pathways. Therefore, in this study we tested whether HMW-MAA-specific monoclonal antibodies (mAb) could inhibit growth of 11q23-positive leukemia cells in SCID mice. Methods  HMW-MAA-specific mAb were tested for their ability to inhibit the in vitro proliferation of an 11q23-positive acute myeloid leukemia (AML) cell line and blasts from four patients with 11q23 aberrations and their in vivo growth in subcutaneous and disseminated xenograft models. Results  The HMW-MAA-specific mAb did not affect in vitro proliferation although they down-regulated phosphorylated (P) Pyk2 expression. Furthermore, the mAb enhanced the in vitro anti-proliferative effect of cytarabine. In vivo the mAb inhibited the growth of leukemic cells in a dose-dependent fashion. However, the difference did not reach statistical significance. No effect was detected on P-Pyk2 expression. Furthermore, HMW-MAA-specific mAb in combination with cytarabine did not improve tumor inhibition. Lastly, the combination of two mAb which recognize distinct HMW-MAA determinants had no detectable effect on survival in a disseminated xenograft model. Conclusions  HMW-MAA-specific mAb down-regulated P-Pyk2 expression and enhanced the anti-proliferative effect of cytarabine in vitro, but had no detectable effect on survival or growth of leukemia cells in vivo. Whether the HMW-MAA-specific mAb can be used as carriers of toxins or chemotherapeutic agents against 11q23-acute leukemia remains to be determined.     

Phage-selected primate antibodies fused to superantigens for immunotherapy of malignant melanoma

 The high-molecular-weight melanoma-associated antigen, HMW-MAA, has been demonstrated to be of potential interest for diagnosis and treatment of malignant melanoma. Murine monoclonal antibodies (mAb) generated in response to different epitopes of this cell-surface molecule efficiently localise to metastatic lesions in patients with disseminated disease. In this work, phage-display-driven selection for melanoma-reactive antibodies generated HMW-MAA specificities capable of targeting bacterial superantigens (SAg) and cytotoxic T cells to melanoma cells. Cynomolgus monkeys were immunised with a crude suspension of metastatic melanoma. A strong serological response towards HMW-MAA demonstrated its role as an immunodominant molecule in the primate. Several clones producing monoclonal scFv antibody fragments that react with HMW-MAA were identified using melanoma cells and tissue sections for phage selection of a recombinant antibody phage library generated from lymph node mRNA. One of these scFv fragments, K305, was transferred and expressed as a Fab-SAg fusion protein and evaluated as the tumour-targeting moiety for superantigen-based immunotherapy. It binds with high affinity to a unique human-specific epitope on the HMW-MAA, and demonstrates more restricted crossreactivity with normal smooth-muscle cells than previously described murine mAb. The K305 Fab was fused to the superantigen staphylococcal enterotoxin A (D227A) [SEA(D227A)], which had been mutated to reduce its intrinsic MHC class II binding affinity, and the fusion protein was used to demonstrate redirection of T cell cytotoxicity to melanoma cells in vitro. In mice with severe combined immunodeficiency, carrying human melanoma tumours, engraftment of human lymphoid cells followed by treatment with the K305Fab-SEA(D227A) fusion protein, induced HMW-MAA-specific tumour growth reduction. The phage-selected K305 antibody demonstrated high-affinity binding and selectivity, supporting its use for tumour therapy in conjunction with T-cell-activating superantigens. Received: 9 September 1999 / Accepted: 21 October 1999     

Human high molecular weight melanoma-associated antigen mimicry by mouse antiidiotypic monoclonal antibody MK2-23. Characterization of the immunogenicity in syngeneic hosts

Previous studies have shown that the mouse antiidiotypic mAb MK2-23 elicited with the syngeneic anti-human high molecular weight melanoma-associated Ag (HMW-MAA) mAb 763.74 elicits anti-HMW-MAA antibodies in syngeneic hosts and in patients with melanoma. The present investigation has characterized the fine specificity of antibodies elicited by mAb MK2-23, tested its ability to induce delayed-type hypersensitivity reaction to HMW-MAA-bearing melanoma cells and analyzed the variables that influence the immunogenicity of mAb MK2-23. The anti-HMW-MAA antibodies elicited by mAb MK2-23 recognize the same population of molecules recognized by mAb 763.74, react with the same (or spatially close) determinant(s) and express the idiotopes recognized by mAb MK2-23 in their Ag-combining sites. The antiidiotypic antibodies that bind to HMW-MAA have a lower titer than those that do not. These results in conjunction with those obtained in mice using a suboptimal immunization schedule suggest that the idiotope(s) that mimic(s) the mAb 763.74-defined determinant of the HMW-MAA is less immunogenic than those that do not. mAb MK2-23 induces a delayed-type hypersensitivity reaction to HMW-MAA-bearing melanoma cells. Therefore, mAb MK2-23 represents the first example of mouse antiidiotypic mAb that induces a cellular and humoral immunity to a human tumor-associated Ag (TAA), because the previously described mouse antiidiotypic mAb that bear the mirror image of TAA have been shown to induce only humoral anti-TAA immunity. The immunogenicity of mAb MK2-23 is markedly enhanced by its conjugation to keyhole limpet hemocyanin and its administration with FA. Furthermore, the number of immunizations and the doses of mAb MK2-23 injected influence its immunogenicity, although to a lower extent than conjugation to a carrier and mixing with an adjuvant. The information derived from the present investigation represents a useful background to optimize the immunization schedule with mAb MK2-23 in patients with melanoma.     

Characterization of syngeneic antiidiotypic monoclonal antibodies to murine anti-human high molecular weight melanoma-associated antigen monoclonal antibodies

Hybridization with murine myeloma cells P3-X63-Ag8.653 of splenocytes from BALB/c mice immunized with syngeneic anti-human high molecular weight melanoma-associated Ag (HMW-MAA) mAb 149.53, 225.28, 763.74, and TP41.2 has resulted in the formation of antiidiotypic antibody-secreting hybridomas with a frequency ranging between 1.2% and 5.2%. No marked difference was detected in the frequency of antibody secreting hybridomas in the fusions generated from mice immunized with the four anti-HMW-MAA mAb, suggesting that the idiotopes expressed by each of them display similar immunogenicity in a syngeneic combination. The number of antiidiotypic mAb that did not inhibit the binding of immunizing mAb to melanoma cells was higher than that of those that died, suggesting that idiotopes not associated with the Ag-combining site are more immunogenic than those that are. The idiotopes recognized by mAb were not detected on a large panel of anti-HLA Class I mAb, anti-HLA Class II mAb, and anti-human melanoma-associated Ag mAb. The latter included also mAb that cross-inhibit the immunizing anti-HMW-MAA mAb. The idiotopes recognized by mAb were not detected on the isolated H and L chain of the immunizing anti-HMW-MAA mAb. Cross-blocking experiments with a selected number of antiidiotypic mAb identified three distinct idiotopes on mAb 149.53, 225.28, and TP41.2 and two on mAb 763.74. Three, 5, 2, and 5 antiidiotypic mAb to idiotopes within the Ag-combining site of mAb 149.53, 225.28, 763.74, and TP41.2, respectively, were tested for their ability to induce anti-HMW-MAA antibodies. Serological and immunochemical assays detected anti-HMW-MAA antibodies only in sera from BALB/c mice immunized with mAb MK2-23. Therefore, mAb MK2-23 can be classified as beta, while the remaining 14 can be classified as gamma.     

High-molecular-weight melanoma-associated antigen mimotope immunizations induce antibodies recognizing melanoma cells

Size and posttranslational modifications are obstacles in the recombinant expression of high-molecular-weight melanoma-associated antigen (HMW-MAA). Creating a tumor antigen mimic via the phage display technology may be a means to overcome this problem for vaccine design. In this study, we aimed to generate an immunogenic epitope mimic of HMW-MAA. Therefore we screened a linear 9mer phage display peptide library, using the anti-HMW-MAA monoclonal antibody (mAb) 225.28S. This antibody mediates antibody-dependent cellular cytotoxicity (ADCC) and has already been used for anti-idiotype therapy trials. Fifteen peptides were selected by mAb 225.28S in the biopanning procedure. They share a consensus sequence, but show only partial homology to the amino acid sequence of the HMW-MAA core protein, indicating mimicry with a conformational epitope. One mimotope was chosen to be fused to albumin binding protein (ABP) as an immunogenic carrier. Immunoassays with 225.28S indicated that the mimotope fusion protein was folded correctly. Subsequently, the fusion protein was tested for immunogenicity in BALB/c mice. The induced anti-mimotope antibodies recognized HMW-MAA of 518A2 human melanoma cells, whereas sera of mice immunized with the carrier ABP alone showed no reactivity. These anti-mimotope antibodies were capable of inducing specific lysis of 518A2 melanoma cells in ADCC assays with murine effector cells. In conclusion, the presented data indicate that mimotopes fused to an immunogenic carrier are suitable tools to elicit epitope-specific anti-melanoma immune responses.     

Efficient killing of tumor cells by CAR-T cells requires greater number of engaged CARs than TCRs

Although CAR-T cells are widely used to treat cancer, efficiency of CAR-T cell cytolytic responses has not been carefully examined. We engineered CAR specific for HMW-MAA (high-molecular-weight melanoma-associated antigen) and evaluated potency of CD8+ CAR-T cells to release cytolytic granules and to kill tissue-derived melanoma cells, which express different levels of HMW-MAA. CAR-T cells efficiently killed melanoma cells expressing high level of HMW-MAA, but not melanoma cells with lower levels of HMW-MAA. The same melanoma cells presenting significantly lower level of stimulatory peptide-MHC ligand were readily lysed by T cells transduced with genes encoding α,β-TCR specific for the peptide-MHC ligand. The data suggest that higher level of targeted molecules is required to engage a larger number of CARs than TCRs to induce efficient cytolytic granule release and destruction of melanoma cells. Understanding the difference in molecular mechanisms controlling activation thresholds of CAR- versus TCR-mediated responses will contribute to improving efficiency of CAR T cells required to eliminate solid tumors presenting low levels of targeted molecules.     

Vaccination of Stage IV patients with allogeneic IL-4- or IL-2-gene-transduced melanoma cells generates functional antibodies against vaccinating and autologous melanoma cells

The antibody (Ab) response to allogeneic Me14932 and autologous melanoma cells was analyzed in 13 Stage IV (AJCC) melanoma patients immunized with Me14932 cells transduced with the IL-4 (Me14932/IL-4) ( n=10) or IL-2 (Me14932/IL-2) ( n=3) gene. No Ab response was observed before the 4th vaccination. Among 8 patients that received four vaccinations, 3/5 patients vaccinated with Me14932/IL-4 cells developed Ab (IgG and/or IgM) to Me14932 ( n=3) and to autologous ( n=2) melanoma cells, and 2/3 patients vaccinated with Me14932/IL-2 cells developed Ab (IgG) to Me14932, but not to autologous melanoma cells. Further, among these 5 responding patients, circulating Ab against the HLA-A3 allele, expressed only on vaccinating cells, were identified in the immune sera of 4 patients immunized with Me14932/IL-4 ( n=2) or Me14932/IL-2 ( n=2) cells. These sera mediated antibody-dependent cell cytotoxicity (ADCC) of Me14932 cells, and a direct correlation ( r=0.85; P=0.03) between intensity of staining (IgG) and extent of lysis was found. Immune serum of one of these patients also induced ADCC of autologous melanoma cells, and serum from another patient mediated complement cytotoxicity of Me14932, but not of autologous melanoma cells. Thus, Abs against vaccinating and autologous melanoma cells were generated in 62% of patients after four vaccinations with cytokine-transduced melanoma cells. These findings demonstrate that the identification and titration of alloreactive Ab helps to monitor the extent of immunization against cellular vaccines, while the induction of Ab reactive to antigens shared between vaccinating and autologous melanoma cells may contribute to their therapeutic efficacy.     

Shared antigens between human malignant melanoma cells and Mycobacterium bovis (BCG).

This study was undertaken to investigate the antigenic relationships between human malignant melanoma cells and Mycobacterium bovis (BCG). Rabbits were immunized with sonicates of BCG or with malignant melanoma cells from different patients and the resulting antisera were tested for their capacity to bind radiolabeled soluble extracts prepared from BCG and melanoma cells. The binding of antibodies to radiolabeled antigens was studied by precipitation of radiolabeled antigen-antibody complexes by anti-rabbit immunoglobulin. Antibodies in sera from rabbits immunized with either BCG (anti-BCG) or melanoma cells (anti-melanoma) bound both the labeled BCG and melanoma antigens. Control antisera, from rabbits immunized with human acute or chronic lymphatic leukemia cells or with normal human spleen cells, did not bind significant amounts of radiolabeled BCG. Antibodies in sera from rabbits immunized with normal spleen cells bound small but significant amounts of radiolabeled melanoma antigens. Binding by anti-BCG and anti-melanoma to the radiolabeled antigens was studied before and after absorption of antisera with cells from human melanoma, leukemia, guinea pig hepatoma, and normal human spleen cells. Inhibition studies using unlabeled BCG extracts also were carried out. The absorption and inhibition studies confirmed that the binding reactions were specific and that antigens from five melanoma patients shared antigenic determinants with BCG.     

Protective immunization against group B meningococci using anti-idiotypic mimics of the capsular polysaccharide

Use of the serogroup B meningococcal capsular polysaccharide (MenB CP) as a vaccine is hampered by the presence of epitopes that cross-react with human polysialic acid. As non-cross-reactive, protective capsular epitopes have also been described, we set out to develop protein mimics of one of such epitopes using as a template a highly protective mAb (mAb Seam 3) raised against a chemically modified form of the MenB CP (N-Pr MenB CP). Using phage display, anti-idiotypic single-chain Ab fragments (scFvs) were obtained from spleen cells of mice immunized with the Seam 3 mAb. Two Seam 3-specific scFvs competed with N-Pr MenB CP for binding to either mAb Seam 3 or rabbit Abs present in typing sera. Moreover, in mice and rabbits the scFvs elicited the production of Abs reacting with both N-Pr MenB CP and whole meningococci, but not with human polysialic acid. These scFv-induced Ab responses were boostable and of the Th1 type, as shown by a predominance of IgG2a. In addition, passive immunization with sera from scFv-immunized animals partially protected neonatal mice from experimental infection with group B meningococci. In conclusion, we have produced anti-idiotypic scFvs that mimic a protective MenB CP epitope and may be useful in the development of an alternative group B meningococcal vaccine.     

Generation of melanoma-specific, cytotoxic CD4(+) T helper 2 cells: requirement of both HLA-DR15 and Fas antigens on melanomas for their lysis by Th2 cells

Recognition of melanoma antigens by HLA class-II-restricted CD4(+) T lymphocytes has been investigated. Two cytotoxic CD4(+) T cell lines were established by stimulating PBLs from a melanoma patient with either parental or IFN-gamma-transduced autologous tumor cells. These T cells secreted IL-4, but not IL-2, IFN-gamma, or TNF-beta, in response to the autologous melanoma cells, suggesting that they belong to the Th2 subtype. Their cytotoxicity was directed against the IFN-gamma-transduced melanoma cells and was HLA-DR-restricted. The autologous and two allogeneic IFN-gamma-modified melanoma cell lines shared melanoma antigen(s) presented in the context of HLA-DR15. HLA-DR15(+) nonmelanoma cells were resistant targets indicating that the shared antigen(s) is melanoma associated. Parental autologous and HLA-DR-matched allogeneic melanoma cell lines, displaying low levels of HLA-DR antigens, induced Th2 proliferation and cytokine release, but were insensitive to lysis prior to upregulation of HLA-DR and Fas antigens by IFN-gamma. Cytolysis was inhibited by anti-HLA-DR and by anti-Fas antibodies, suggesting that the cytolysis is mediated via the Fas pathway. While small amounts of HLA-DR15 molecules on melanoma cells are sufficient for Th2 proliferation and cytokine release, higher amounts of HLA-DR15 and the expression of Fas are required for CD4(+)-mediated lysis.     

Regression of human melanoma xenografts in nude mice injected with methotrexate linked to monoclonal antibody 225.28 to human high molecular weight-melanoma associated antigen

Summary Intravenous injections into nude mice of 5 mg/kg methotrexate (MTX) linked to the antibody to human high molecular weight-melanoma associated antigen (HMW-MAA), monoclonal antibody (mAb) 225.28, an IgG_2a, on days 1, 4, 7, 10 and 14, starting 24 h after subcutaneous inoculation of 2 × 10⁶ cultured human M21 melanoma cells inhibited mean tumor volume by 90% on day 14 and by 65% on day 50 after the beginning of the treatment. Injections of equimolar amounts of free MTX and MTX linked to normal mouse IgG or to an isotypematched myeloma protein did not inhibit tumor growth significantly. MTX linked to mAb 225.28 did not inhibit the xenograft of a subline of human melanoma cell line M21 without detectable expression of HMW-MAA. In a clonogenic assay, the MTX-225.28 conjugate was three times more potent in inhibiting the growth of M21 melanoma cells than free MTX, but did not inhibit the growth of kidney carcinoma cells Caki-1, which do not express high-M _r MAA. In contrast, MTX linked to the mAb DAL K29, reacting with kidney carcinoma cells Caki-1, inhibited their growth but did not affect that of melanoma cells. M21 melanoma cells isolated from the residual tumor of a mouse treated with the MTX-225.28 conjugate did not differ in their reactivity with mAb 225.28 and in their sensitivity to MTX when compared with M21 cells from an untreated mouse.     

Selective targeting of melanoma and APCs using a recombinant antibody with TCR-like specificity directed toward a melanoma differentiation antigen

Tumor-associated, MHC-restricted peptides, recognized by tumor-specific CD8(+) lymphocytes, are desirable targets for novel approaches in immunotherapy because of their highly restricted fine specificity. Abs that recognize these tumor-associated MHC-peptide complexes, with the same specificity as TCR, would therefore be valuable reagents for studying Ag presentation by tumor cells, for visualizing MHC-peptide complexes on cells, and eventually for developing new targeting agents for cancer immunotherapy. To generate molecules with such a unique, fine specificity, we immunized HLA-A2 transgenic mice with a single-chain HLA-A2, complexed with a common antigenic T cell HLA-A2-restricted epitope derived from the melanoma differentiation Ag gp100. Using a phage display approach, we isolated a recombinant scFv Ab that exhibits a characteristic TCR-like binding specificity, yet, unlike TCRs, it did so with a high affinity in the nanomolar range. The TCR-like Ab can recognize the native MHC-peptide complex expressed on the surface of APCs, and on peptide-pulsed or native melanoma cells. Moreover, when fused to a very potent cytotoxic effector molecule in the form of a truncated bacterial toxin, it was able to specifically kill APCs in a peptide-dependent manner. These results demonstrate the utility of high affinity TRC-like scFv recombinant Abs directed toward human cancer T cell epitopes. Such TCR-like Abs may prove to be very useful for monitoring and visualizing the expression of specific MHC-peptide complexes on the surface of tumor cells, APCs, and lymphoid tissues, as well as for developing a new family of targeting agents for immunotherapy.     

Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by alpha-galactosylceramide in mice

The role of mouse liver NK1.1 Ag(+) T (NKT) cells in the antitumor effect of alpha-galactosylceramide (alpha-GalCer) has been unclear. We now show that, whereas alpha-GalCer increased the serum IFN-gamma concentration and alanine aminotransferase activity in NK cell-depleted C57BL/6 (B6) mice and B6-beige/beige mice similarly to its effects in control B6 mice, its enhancement of the antitumor cytotoxicity of liver mononuclear cells (MNCs) was abrogated. Depletion of both NK and NKT cells in B6 mice reduced all these effects of alpha-GALCER: Injection of Abs to IFN-gamma also inhibited the alpha-GalCer-induced increase in antitumor cytotoxicity of MNCS: alpha-GalCer induced the expression of Fas ligand on NKT cells in the liver of B6 mice. Whereas alpha-GalCer did not increase serum alanine aminotransferase activity in B6-lpr/lpr mice and B6-gld/gld mice, it increased the antitumor cytotoxicity of liver MNCS: The alpha-GalCer-induced increase in survival rate apparent in B6 mice injected intrasplenically with B16 tumor cells was abrogated in beige/beige mice, NK cell-depleted B6 mice, and B6 mice treated with Abs to IFN-gamma. Depletion of CD8(+) T cells did not affect the alpha-GalCer-induced antitumor cytotoxicity of liver MNCs but reduced the effect of alpha-GalCer on the survival of B6 mice. Thus, IFN-gamma produced by alpha-GalCer-activated NKT cells increases both the innate antitumor cytotoxicity of NK cells and the adaptive antitumor response of CD8(+) T cells, with consequent inhibition of tumor metastasis to the liver. Moreover, NKT cells mediate alpha-GalCer-induced hepatocyte injury through Fas-Fas ligand signaling.     

Antibodies against RNA hydrolyze RNA and DNA

Immunization of animals with DNA leads to the production of anti-DNA antibodies (Abs) demonstrating both DNase and RNase activities. It is currently not known whether anti-RNA Abs can possess nuclease activities. In an attempt to address this question, we have shown that immunization of three rabbits with complex of RNA with methylated BSA (mBSA) stimulates production of IgGs with RNase and DNase activities belonging to IgGs, while polyclonal Abs from three non-immunized rabbits and three animals immunized with mBSA are catalytically inactive. Affinity chromatography of IgGs from the sera of autoimmune (AI) patients on DNA-cellulose usually demonstrates a number of fractions, all of which effectively hydrolyze both DNA and RNA, while rabbit catalytic IgGs were separated into Ab subfractions, some of which demonstrated only DNase activity, while others hydrolyzed RNA faster than DNA. The enzymic properties of the RNase and DNase IgGs from rabbits immunized with RNA distinguish them from all known canonical RNases and DNases and DNA- and RNA-hydrolyzing abzymes (Abzs) from patients with different AI diseases. In contrast to RNases and AI RNA-hydrolyzing Abs, rabbit RNase IgGs catalyze only the first step of the hydrolysis reaction but cannot hydrolyze the formed terminal 2',3'-cyclophosphate. The data indicate that Abzs of AI patients hydrolyzing nucleic acids in part may be Abs against RNA and its complexes with proteins. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Effective mucosal immunity to anthrax: neutralizing antibodies and Th cell responses following nasal immunization with protective antigen

Mucosal, but not parenteral, immunization induces immune responses in both systemic and secretory immune compartments. Thus, despite the reports that Abs to the protective Ag of anthrax (PA) have both anti-toxin and anti-spore activities, a vaccine administered parenterally, such as the aluminum-adsorbed anthrax vaccine, will most likely not induce the needed mucosal immunity to efficiently protect the initial site of infection with inhaled anthrax spores. We therefore took a nasal anthrax vaccine approach to attempt to induce protective immunity both at mucosal surfaces and in the peripheral immune compartment. Mice nasally immunized with recombinant PA (rPA) and cholera toxin (CT) as mucosal adjuvant developed high plasma PA-specific IgG Ab responses. Plasma IgA Abs as well as secretory IgA anti-PA Abs in saliva, nasal washes, and fecal extracts were also induced when a higher dose of rPA was used. The anti-PA IgG subclass responses to nasal rPA plus CT consisted of IgG1 and IgG2b Abs. A more balanced profile of IgG subclasses with IgG1, IgG2a, and IgG2b Abs was seen when rPA was given with a CpG oligodeoxynucleotide as adjuvant, suggesting a role for the adjuvants in the nasal rPA-induced immunity. The PA-specific CD4(+) T cells from mice nasally immunized with rPA and CT as adjuvant secreted low levels of CD4(+) Th1-type cytokines in vitro, but exhibited elevated IL-4, IL-5, IL-6, and IL-10 responses. The functional significance of the anti-PA Ab responses was established in an in vitro macrophage toxicity assay in which both plasma and mucosal secretions neutralized the lethal effects of Bacillus anthracis toxin.