Anti-NeuGcGM3 antibodies, actively elicited by idiotypic vaccination in nonsmall cell lung cancer patients, induce tumor cell death by an oncosis-like mechanism

1E10 is a murine anti-idiotypic mAb specific for an idiotypic mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In melanoma, breast, and lung cancer patients, this anti-idiotypic Ab was able to induce a specific Ab response against N-glycosylated gangliosides, attractive targets for cancer immunotherapy as these glycolipids are not naturally expressed in humans. A clinical study with nonsmall cell lung cancer patients showed encouraging clinical benefits. Immunological studies performed in 20 of these patients suggested a correlation between the induction of Abs against NeuGcGM3 and longer survival times. The induced anti-NeuGcGM3 Abs recognized and directly killed tumor cells expressing the Ag, by a mechanism independent of complement activation. In the present work, we show that this cytotoxicity differs from apoptosis because it is temperature independent, no chromatin condensation or caspase 3 induction are detected, and the DNA fragmentation induced has a different pattern than the one characteristic for apoptosis. It is a very quick process and involves cytosqeleton reorganization. The Abs induce cellular swelling and the formation of big membrane lesions that allow the leakage of cytoplasm and the loss of the cell membrane integrity. All of these characteristics resemble a process of oncotic necrosis. To our knowledge, this is the first report of the active induction in cancer patients of NeuGcGM3-specific Abs able to induce complement independent oncotic necrosis to tumor cells. These results contribute to reinforcing the therapeutic potential of anti-idiotypic vaccines and the importance of NeuGcGM3 ganglioside as antitumor target.     

Recombinant viral-vectored vaccines expressing Plasmodium chabaudi AS apical membrane antigen 1: mechanisms of vaccine-induced blood-stage protection

Apical membrane Ag 1 (AMA1) is one of the leading candidate Ags for inclusion in a subunit vaccine against blood-stage malaria. However, the efficacy of Ab-inducing recombinant AMA1 protein vaccines in phase IIa/b clinical trials remains disappointing. In this article, we describe the development of recombinant human adenovirus serotype 5 and modified vaccinia virus Ankara vectors encoding AMA1 from the Plasmodium chabaudi chabaudi strain AS. These vectors, when used in a heterologous prime-boost regimen in BALB/c mice, are capable of inducing strong transgene-specific humoral and cellular immune responses. We show that this vaccination regimen is protective against a nonlethal P. chabaudi chabaudi strain AS blood-stage challenge, resulting in reduced peak parasitemias. The role of vaccine-induced, AMA1-specific Abs and T cells in mediating the antiparasite effect was investigated by in vivo depletion of CD4(+) T cells and adoptive-transfer studies into naive and immunodeficient mice. Depletion of CD4(+) T cells led to a loss of vaccine-induced protection. Adoptive-transfer studies confirmed that efficacy is mediated by both CD4(+) T cells and Abs functioning in the context of an intact immune system. Unlike previous studies, these results confirm that Ag-specific CD4(+) T cells, induced by a clinically relevant vaccine-delivery platform, can make a significant contribution to vaccine blood-stage efficacy in the P. chabaudi model. Given that cell-mediated immunity may also contribute to parasite control in human malaria, these data support the clinical development of viral-vectored vaccines that induce both T cell and Abs against Plasmodium falciparum blood-stage malaria Ags like AMA1.     

Selection of GM2, fucosyl GM1, globo H and polysialic acid as targets on small cell lung cancers for antibody mediated immunotherapy

Glycolipids GM2, GD2, GD3, fucosyl GM1, sialyl Lewis a (sLe^a) and globo H, and polysialic acid on embryonal NCAM, are cell-surface antigens expressed on small cell lung cancer (SCLC) biopsy specimens. They are all candidates for inclusion in a polyvalent, antibody-inducing vaccine or for adoptive therapy with monoclonal antibodies (mAbs) against SCLC. To identify the minimum optimal combination of target antigens on SCLC and to confirm that antibodies against this combination might be able to mediate complement activation and lysis in the majority of cases, we tested ten SCLC cell lines with fluorescence activated cell sorter (FACS) and complement dependent cytotoxicity (CDC) assays using mAbs against these seven target antigens individually or pooled in different combinations. We find that (1) none of these mAbs demonstrated strong FACS reactivity with more than 6 of the 10 cell lines, (2) no mAb had strong CDC reactivity with more than 4 of the cell lines, (3) when the mAbs were pooled, nine cell lines were strongly positive by FACS and nine cell lines were strongly positive by CDC, and (4) mAbs against GM2, FucGM1, globo H and polysialic acid was the minimum optimal combination for inducing FACS reactivity. The addition of mAbs against sLe^a, GD2 and GD3 had no additional impact by FACS and only minimal additional impact in CDC assays. H345, the only cell line that had less than 30% CDC with the four mAb pool was strongly positive by FACS. To understand the lack of correlation between FACS and CDC in the case of H345, the ten cell lines were screened for expression of complement resistance factors CD55 and CD59. Three cell lines were strongly positive for CD55 and eight were strongly positive for CD59. Overall, no correlation was seen between expression of either of these factors on the ten cell lines and sensitivity to CDC. In the case of H345 however, complement resistance of H345 is demonstrated to be mediated primarily by CD59, and in the presence of mAb against CD59, the four mAb MEM-43 pool induced strong (94%) CDC. CD59 inhibits membrane attack complex formation but not activation of earlier complement components. Consequently, all ten cell lines are good targets for complement activation by the four antibody pool and for elimination by effector mechanisms including complement mediated inflammation and opsonization. These findings support our plan to develop a tetravalent vaccine against SCLC targeting GM2, fucosyl GM1, globo H and polysialic acid.Supported by grants from the NIH (PO1CA33049), and the Lawrence and Selma Ruben Foundations.     

Enhancing blood-stage malaria subunit vaccine immunogenicity in rhesus macaques by combining adenovirus, poxvirus, and protein-in-adjuvant vaccines

Protein-in-adjuvant formulations and viral-vectored vaccines encoding blood-stage malaria Ags have shown efficacy in rodent malaria models and in vitro assays against Plasmodium falciparum. Abs and CD4(+) T cell responses are associated with protective efficacy against blood-stage malaria, whereas CD8(+) T cells against some classical blood-stage Ags can also have a protective effect against liver-stage parasites. No subunit vaccine strategy alone has generated demonstrable high-level efficacy against blood-stage infection in clinical trials. The induction of high-level Ab responses, as well as potent T and B cell effector and memory populations, is likely to be essential to achieve immediate and sustained protective efficacy in humans. This study describes in detail the immunogenicity of vaccines against P. falciparum apical membrane Ag 1 in rhesus macaques (Macaca mulatta), including the chimpanzee adenovirus 63 (AdCh63), the poxvirus modified vaccinia virus Ankara (MVA), and protein vaccines formulated in Alhydrogel or CoVaccine HT adjuvants. AdCh63-MVA heterologous prime-boost immunization induces strong and long-lasting multifunctional CD8(+) and CD4(+) T cell responses that exhibit a central memory-like phenotype. Three-shot (AdCh63-MVA-protein) or two-shot (AdCh63-protein) regimens induce memory B cells and high-titer functional IgG responses that inhibit the growth of two divergent strains of P. falciparum in vitro. Prior immunization with adenoviral vectors of alternative human or simian serotype does not affect the immunogenicity of the AdCh63 apical membrane Ag 1 vaccine. These data encourage the further clinical development and coadministration of protein and viral vector vaccine platforms in an attempt to induce broad cellular and humoral immune responses against blood-stage malaria Ags in humans.     

Hybrids of dendritic cells and tumor cells generated by electrofusion simultaneously present immunodominant epitopes from multiple human tumor-associated antigens in the context of MHC class I and class II molecules

Hybrid cells generated by fusing dendritic cells with tumor cells (DC-TC) are currently being evaluated as cancer vaccines in preclinical models and human immunization trials. In this study, we evaluated the production of human DC-TC hybrids using an electrofusion protocol previously defined for murine cells. Human DCs were electrically fused with allogeneic melanoma cells (888mel) and were subsequently analyzed for coexpression of unique DC and TC markers using FACS and fluorescence microscopy. Dually fluorescent cells were clearly observed using both techniques after staining with Abs against distinct surface molecules suggesting that true cell fusion had occurred. We also evaluated the ability of human DC-TC hybrids to present tumor-associated epitopes in the context of both MHC class I and class II molecules. Allogeneic DCs expressing HLA-A*0201, HLA-DR beta 1*0401, and HLA-DR beta 1*0701 were fused with 888mel cells that do not express any of these MHC molecules, but do express multiple melanoma-associated Ags. DC-888mel hybrids efficiently presented HLA-A*0201-restricted epitopes from the melanoma Ags MART-1, gp100, tyrosinase, and tyrosinase-related protein 2 as evaluated by specific cytokine secretion from six distinct CTL lines. In contrast, DCs could not cross-present MHC class I-restricted epitopes after exogenously loading with gp100 protein. DC-888mel hybrids also presented HLA-DR beta 1*0401- and HLA-DR beta 1*0701-restricted peptides from gp100 to CD4(+) T cell populations. Therefore, fusions of DCs and tumor cells express both MHC class I- and class II-restricted tumor-associated epitopes and may be useful for the induction of tumor-reactive CD8(+) and CD4(+) T cells in vitro and in human vaccination trials.     

The autoreactivity of anti-phosphorylcholine antibodies for atherosclerosis-associated neo-antigens and apoptotic cells

Abs specific for phosphorylcholine (PC) are known to contribute to the immune defense against a variety of microbial infections. To assess for other types of binding interactions, we performed surveys of anti-PC Abs of diverse biologic origins and structural diversity and demonstrated a common autoreactivity for oxidatively modified low density lipoprotein and other oxidation-specific structures containing PC-Ags. We also found that cells undergoing apoptosis sequentially express a range of oxidation-specific neo-self PC determinants. Whereas natural Abs to PC recognized cells at early stages of apoptosis, by contrast, an IgG anti-PC Ab, representative of a T cell-dependent response, recognized PC determinants primarily associated with late stages of apoptosis. Cumulatively, these results demonstrate a fundamental paradigm in which Abs from both the innate and the T cell-dependent tiers of the B cell compartment recognize a minimal molecular motif arrayed both on microbes and as neo-self Ags linked to atherosclerosis and autoimmune disease.     

Identification of protective B cell antigens of Legionella pneumophila

Abs confer protection from secondary infection with Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaires' disease. In this study, we demonstrate that Ab-mediated protection is effective across L. pneumophila serogroups, suggesting that Abs specific for conserved protein Ags are sufficient to mediate this protective effect. We used two independent methods to identify immunogenic L. pneumophila protein Ags, namely, the screening of a λ phage library representing the complete L. pneumophila genome and two-dimensional gel electrophoresis combined with Western blot analysis and protein spot identification by mass spectrometry. A total of 30 novel L. pneumophila B cell Ags were identified, the majority of which are located in or associated with the bacterial membrane, where they are accessible for Abs and, therefore, likely to be relevant for Ab-mediated protection against L. pneumophila. Selected B cell Ags were recombinantly expressed and tested in a vaccination protocol. Mice immunized with either single-protein Ags or an Ag combination showed reduced bacterial titers in bronchoalveolar lavage and lung after L. pneumophila challenge. To determine the clinical relevance of these findings, we tested Legionnaires' disease patient sera for reactivity with the identified L. pneumophila Ags. The recognized Ags were indeed conserved across host species, because Abs specific for all three selected Ags could be detected in patient sera, rendering the identified protein Ags potential vaccine candidates.     

Novel blocking human IgG directed against the pentapeptide repeat motifs of Neisseria meningitidis Lip/H.8 and Laz lipoproteins

Ab-initiated, complement-dependent killing contributes to host defenses against invasive meningococcal disease. Sera from nonimmunized individuals vary widely in their bactericidal activity against group B meningococci. We show that IgG isolated from select individuals can block killing of group B meningococci by human sera that are otherwise bactericidal. This IgG also reduced the bactericidal efficacy of Abs directed against the group B meningococcal protein vaccine candidates factor H-binding protein currently undergoing clinical trials and Neisserial surface protein A. Immunoblots revealed that the blocking IgG was directed against a meningococcal Ag called H.8. Killing of meningococci in reactions containing bactericidal mAbs and human blocking Abs was restored when binding of blocking Ab to meningococci was inhibited using either synthetic peptides corresponding to H.8 or a nonblocking mAb against H.8. Furthermore, genetic deletion of H.8 from target organisms abrogated blocking. The Fc region of the blocking IgG was required for blocking because F(ab')(2) fragments were ineffective. Blocking required IgG glycosylation because deglycosylation with peptide:N-glycanase eliminated blocking. C4b deposition mediated by an anti-factor H-binding protein mAb was reduced by intact blocking IgG, but not by peptide:N-glycanase-treated blocking IgG, suggesting that blocking resulted from inhibition of classical pathway of complement. In conclusion, we have identified H.8 as a meningococcal target for novel blocking Abs in human serum. Such blocking Abs may reduce the efficacy of select antigroup B meningococcal protein vaccines. We also propose that outer membrane vesicle-containing meningococcal vaccines may be more efficacious if purged of subversive immunogens such as H.8.     

IL-4 and T cells are required for the generation of IgG1 isotype antibodies against cardiolipin

Infection with Mycobacterium tuberculosis induces Abs against a vast array of mycobacterial lipids and glycolipids. One of the most prominent lipid Ags recognized is cardiolipin (CL). The kinetics of the generation of anti-CL Abs during infection reveals that IgM titers to CL increase over time. Interestingly, at day 30 postinfection CL-specific IgG1 appears, an isotype usually dependent on T cell help. Using an immunization schedule with CL/anti-CL Ab complexes, which induces antiphospholipid syndrome in mice, we show that the generation of IgG1 to CL requires IL-4 and that optimal production is T cell dependent. IgG1 production to CL was impaired in nude (nu/nu) mice devoid in conventional T cells, but was not affected in mice deficient for either alphabeta TCR(+), gammadelta TCR(+), CD4(+), CD8(+), or NK1.1(+) T cells. We conclude that IgG1 production to CL depends on T cell help and IL-4, which can be provided by different T cell populations. This is the first report that IL-4 is indispensable for the induction of IgG1 Abs to lipid Ags.     

Antibodies to keyhole limpet hemocyanin cross-react with an epitope on the polysaccharide capsule of Cryptococcus neoformans and other carbohydrates: implications for vaccine development

Cryptococcus neoformans causes a life-threatening meningoencephalitis in AIDS patients. Mice immunized with a glycoconjugate vaccine composed of the glucuronoxylomannan (GXM) component of the cryptococcal capsular polysaccharide conjugated to tetanus toxoid produce Abs that can be either protective or nonprotective. Because nonprotective Abs block the efficacy of protective Abs, an effective vaccine must focus the Ab response on a protective epitope. Mice immunized with peptide mimetics of GXM conjugated to keyhole limpet hemocyanin (KLH) with glutaraldehyde developed Abs to GXM. However, control peptides P315 and P24 conjugated to KLH also elicited Abs to GXM. GXM-binding Abs from mice immunized with P315-KLH were inhibited by KLH treated with glutaraldehyde (KLH-g), but not by P315. Furthermore, KLH-g inhibited binding of GXM by serum of mice immunized with GXM-TT, indicating that glutaraldehyde treatment of KLH reveals an epitope(s) that cross-reacts with GXM. Vaccination with KLH-g or unmodified KLH elicited Abs to GXM, but did not confer protection against C. neoformans, suggesting the cross-reactive epitope on KLH was not protective. This was supported by the finding that 4H3, a nonprotective mAb, cross-reacted strongly with KLH-g. Sera from mice immunized with either native KLH or KLH-g cross-reacted with several other carbohydrate Ags, many of which have been conjugated to KLH for vaccine development. This study illustrates how mAbs can be used to determine the efficacy of potential vaccines, in addition to describing the complexity of using KLH and glutaraldehyde in the development of vaccines to carbohydrate Ags.     

Comparison of antigen constructs and carrier molecules for augmenting the immunogenicity of the monosaccharide epithelial cancer antigen Tn

We have demonstrated previously that the optimal method for inducing an antibody response against defined cancer antigens is covalent conjugation of the antigen to keyhole limpet hemocyanin (KLH) and use of the potent saponin adjuvant QS-21. Single molecules of glycolipids (tetrasaccharides, pentasaccharides, or hexasaccharides) and MUC1 peptides (containing between one and five MUC1 tandem repeats) conjugated to KLH have proven sufficient for antibody recognition and vaccine construction. However, cancer specificity of monoclonal antibodies against the monosaccharide Tn and disaccharide sTn comes largely from recognition of clusters (c) of these molecules on the cell surface. Tn consists of a monosaccharide (GalNAc) O-linked to serine or threonine on epithelial cancer mucins which are uniquely rich in serines and threonines. We test here several Tn constructs: Tn monosaccharide, Tn(c) prepared on a triple threonine backbone, and Tn prepared on a partially or fully glycosylated MUC1 backbone. We determine that Tn(c) is more effective than Tn, and conjugation to KLH is more effective than conjugation to BSA or polystyrene beads for inducing ELISA reactivity against Tn, and FACS reactivity against Tn-positive tumor cells. Surprisingly, MUC1 glycosylated with Tn at three or five sites per 20 amino acid MUC1 tandem repeat and conjugated to KLH, induced the strongest antibody response against Tn and tumor cells expressing Tn, and had the additional advantage of inducing antibodies against MUC1.     

Generation of a complement-independent bactericidal IgM against a relapsing fever Borrelia

The spirochetemia of relapsing fever in mice is cleared by a complement-independent, polyclonal IgM response with reactivity to two prominent Ags of 20 and 35 kDa. In this study, we have dissected the polyclonal IgM Ab response against a relapsing fever spirochete to determine the specificity of its complement-independent bactericidal properties. Our experimental approach selectively generated an IgM murine mAb from the early specific immune response to a variable outer membrane protein. This IgM is bactericidal in the absence of complement and is part of the polyclonal Ab response that mediates the clearance of this bacterium from the blood. Purified monoclonal IgM caused direct structural damage to the outer membrane of the spirochete, in the absence of complement, and protected both B cell- and C5-deficient mice from challenge when administered passively. The direct, complement-independent, bactericidal activity of Abs is a critical mechanism of host defense against infection.     

Human male genital tract secretions: both mucosal and systemic immune compartments contribute to the humoral immunity

In contrast to numerous studies of female genital tract secretions, the molecular properties of Abs and the magnitude of humoral responses in human male genital tract secretions to naturally occurring Ags and to mucosal and systemic immunizations have not been extensively investigated. Therefore, seminal plasma (SP) collected from healthy individuals was analyzed with respect to Ig levels, their isotypes, molecular forms of IgA, and for the presence of Abs to naturally occurring Ags, or induced by systemic or mucosal immunizations with viral and bacterial vaccines. The results indicated that in SP, IgG and not IgA, is the dominant Ig isotype, and that IgM is present at low levels. IgA is represented by secretory IgA, polymeric IgA, and monomeric IgA. In contrast to the female genital tract secretions in which IgA2 occurs in slight excess, the distribution of IgA subclasses in SP resembles that in plasma with a pronounced preponderance of IgA1. The IgG subclass profiles in SP are also similar to those in serum. Thus, SP is an external secretion that shares common features with both typical external secretions and plasma. Specifically, SP contains naturally occurring secretory IgA Abs to environmental Ags of microbial origin and to an orally administered bacterial vaccine, and plasma-derived IgG Abs to systemically injected vaccines. Therefore, both mucosal and systemic immunization with various types of Ags can induce humoral responses in SP. These findings should be considered in immunization strategies to induce humoral responses against sexually transmitted infections, including HIV-1.     

Optimizing the efficacy of epitope-directed DNA vaccination

An increasing number of clinical trials has been initiated to test the potential of prophylactic or curative vaccination with tumor Ag-encoding DNA vaccines. However, in the past years it has become apparent that for many Ags and in particular for tumor Ags the intracellular processing and presentation are suboptimal. To improve epitope-directed DNA vaccines we have developed a murine model system in which epitope-specific, DNA vaccine-induced T cell immunity can be followed by MHC tetramer technology directly ex vivo. We have used this well-defined model to dissect the parameters that are crucial for the induction of strong cytotoxic T cell immunity using two independent model Ags. These experiments have led to a set of five guidelines for the design of epitope-directed DNA vaccines, indicating that carboxyl-terminal fusion of the epitope to a carrier protein of foreign origin is the most favorable strategy. DNA vaccines that are based on these guidelines induce high-magnitude CD8(+) T cell responses in >95% of vaccinated animals. Moreover, T cell immunity induced by this type of optimized DNA vaccine provides long-term protection against otherwise lethal tumor challenges.     

No advantage of cell-penetrating peptides over receptor-specific antibodies in targeting antigen to human dendritic cells for cross-presentation

Induction of CTL responses by dendritic cell (DC)-based vaccines requires efficient DC-loading strategies for class I Ags. Coupling Ags to cell-penetrating peptides (CPPs) or receptor-specific Abs improves Ag loading of DCs. In contrast to CPPs, receptor-specific Abs deliver conjugated Ags to DCs with high specificity, which is advantageous for in vivo strategies. It has, however, been speculated that CPPs facilitate uptake and endosomal escape of conjugated Ags, which would potently enhance cross-presentation. In this study, we directly compare the in vitro targeting efficiency of a humanized D1 Ab directed against the human DC surface receptor DC-SIGN hD1 to that of three CPPs. The three CPPs colocalized within endosomes when targeted to human monocyte-derived DCs simultaneously, whereas hD1 was present in a different set of endosomes. However, within 75 min after uptake CPPs and hD1 colocalized extensively within the lysosomal compartment. Ab-mediated targeting of class I-restricted peptides to DC-SIGN enhanced cross-presentation of the peptides, while only one of the CPPs enhanced peptide presentation. This CPP and hD1 enhanced cross-presentation with equal efficiencies. Thus, we found no evidence of CPP specifically favoring the delivery of conjugated Ag to the DC class I presentation pathway. Given the specificity with which Abs recognize their targets, this favors the use of DC receptor-specific Abs for in vivo vaccination strategies.     

Antibody-targeted NY-ESO-1 to mannose receptor or DEC-205 in vitro elicits dual human CD8+ and CD4+ T cell responses with broad antigen specificity

Immunization of cancer patients with vaccines containing full-length tumor Ags aims to elicit specific Abs and both CD4(+) and CD8(+) T cells. Vaccination with protein Ags, however, often elicits only CD4(+) T cell responses without inducing Ag-specific CD8(+) T cells, as exogenous protein is primarily presented to CD4(+) T cells. Recent data revealed that Ab-mediated targeting of protein Ags to cell surface receptors on dendritic cells could enhance the induction of both CD4(+) and CD8(+) T cells. We investigated in this study if these observations were applicable to NY-ESO-1, a cancer-testis Ag widely used in clinical cancer vaccine trials. We generated two novel targeting proteins consisting of the full-length NY-ESO-1 fused to the C terminus of two human mAbs against the human mannose receptor and DEC-205, both internalizing molecules expressed on APC. These targeting proteins were evaluated for their ability to activate NY-ESO-1-specific human CD4(+) and CD8(+) T cells in vitro. Both targeted NY-ESO-1 proteins rapidly bound to their respective targets on APC. Whereas nontargeted and Ab-targeted NY-ESO-1 proteins similarly activated CD4(+) T cells, cross-presentation to CD8(+) T cells was only efficiently induced by targeted NY-ESO-1. In addition, both mannose receptor and DEC-205 targeting elicited specific CD4(+) and CD8(+) T cells from PBLs of cancer patients. Receptor-specific delivery of NY-ESO-1 to APC appears to be a promising vaccination strategy to efficiently generate integrated and broad Ag-specific immune responses against NY-ESO-1 in cancer patients.     

Complement is essential for protection by an IgM and an IgG3 monoclonal antibody against experimental, hematogenously disseminated candidiasis

The incidence of life-threatening, hematogenously disseminated candidiasis, which is predominantly caused by Candida albicans, parallels the use of modern medical procedures that adversely affect the immune system. Limited antifungal drug choices and emergence of drug-resistant C. albicans strains indicate the need for novel prevention and therapeutic strategies. We are developing vaccines and Abs that enhance resistance against experimental candidiasis. However, the prevalence of serum anti-Candida Abs in candidiasis patients has led to the misconception that Abs are not protective. To explain the apparent discrepancy between such clinical observations and our work, we compared functional activities of C. albicans-specific protective and nonprotective mAbs. Both kinds of Abs are agglutinins that fix complement and are specific for cell surface mannan, but the protective Abs recognize beta-mannan, and the nonprotective Ab is specific for alpha-mannan. By several indirect and direct measures, the protective mAbs more efficiently bind complement factor C3 to the yeast cell than do nonprotective Ab. We hypothesize that the C3 deposition causes preferential association of blood-borne fungi with host phagocytic cells that are capable of killing the fungus. We conclude from these results that the protective potential of Abs is dependent on epitope specificity, serum titer, and ability to rapidly and efficiently fix complement to the fungal surface. The mechanism of protection appears to be associated with enhanced phagocytosis and killing of the fungus.     

Intratumoral injection of alpha-gal glycolipids induces xenograft-like destruction and conversion of lesions into endogenous vaccines

This study describes a novel cancer immunotherapy treatment that exploits the natural anti-Gal Ab to destroy tumor lesions and convert them into an endogenous vaccine targeted to APC via FcgammaR. Anti-Gal constitutes 1% of immunoglobulins in humans and interacts specifically with alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R). The binding of anti-Gal to alpha-gal epitopes on pig cells mediates xenograft rejection. The proposed method uses glycolipid micelles with multiple alpha-gal epitopes (alpha-gal glycolipids). These glycolipids are extracted from rabbit red cell membranes and are comprised of ceramides with carbohydrate chains containing 5-25 carbohydrates, all capped with alpha-gal epitopes. Efficacy of this treatment was demonstrated in alpha1,3-galactosyltransferase knockout mice producing anti-Gal and bearing B16 melanoma or B16/OVA producing OVA as a surrogate tumor Ag. These mice are unique among nonprimate mammals in that, similar to humans, they lack alpha-gal epitopes and can produce the anti-Gal Ab. Intratumoral injection of alpha-gal glycolipids results in local inflammation mediated by anti-Gal binding to the multiple alpha-gal epitopes and activation of complement. These glycolipids spontaneously insert into tumor cell membranes. The binding of anti-Gal to alpha-gal expressing tumor cells induces the destruction of treated lesions as in anti-Gal-mediated xenograft rejection. Anti-Gal further opsonizes tumor cells within the lesion and, thus, targets them for effective uptake by APC that transport the tumor Ags to draining lymph nodes. APC further cross-present immunogenic tumor Ag peptides and elicit a systemic anti-tumor immune response. Similar intratumoral injection of alpha-gal glycolipids in humans is likely to induce the destruction of treated lesions and elicit a protective immune response against micrometastases.     

Intracellular domains of target antigens influence their capacity to trigger antibody-dependent cell-mediated cytotoxicity

Ab-mediated signaling in tumor cells and Ab-dependent cell-mediated cytotoxicity (ADCC) are both considered as relevant effector mechanisms for Abs in tumor therapy. To address potential interactions between these two mechanisms, we generated HER-2/neu- and CD19-derived chimeric target Ags, which were expressed in experimental tumor target cells. HER-2/neu-directed Abs were documented to mediate effective ADCC with both mononuclear cells (MNCs) and polymorphonuclear granulocytes (PMNs), whereas Abs against CD19 were effective only with MNCs and not with PMNs. We generated cDNA encoding HER-2/CD19 or CD19/HER-2 (extracellular/intracellular) chimeric fusion proteins by combining cDNA encoding extracellular domains of HER-2/neu or CD19 with intracellular domains of CD19 or HER-2/neu, respectively. After transfecting wild-type HER-2/neu or chimeric HER-2/CD19 into Raji Burkitt's lymphoma cells and wild-type CD19 or chimeric CD19/HER-2 into SK-BR-3 breast cancer cells, target cell lines were selected for high membrane expression of transfected Ags. We then investigated the efficacy of tumor cell lysis by PMNs or MNCs with CD19- or HER-2/neu-directed Ab constructs. MNCs triggered effective ADCC against target cells expressing wild-type or chimeric target Ag. As expected, PMNs killed wild-type HER-2/neu-transfected, but not wild-type CD19-transfected target cells. Interestingly, however, PMNs were also effective against chimeric CD19/HER-2-transfected, but not HER-2/CD19-transfected target cells. In conclusion, these results demonstrate that intracellular domains of target Ags contribute substantially to effective Ab-mediated tumor cell killing by PMNs.     

Intratumoral injection of α-gal glycolipids induces a protective anti-tumor T cell response which overcomes Treg activity

α-Gal glycolipids capable of converting tumors into endogenous vaccines, have α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R) and are extracted from rabbit RBC membranes. α-Gal epitopes bind anti-Gal, the most abundant natural antibody in humans constituting 1% of immunoglobulins. α-Gal glycolipids insert into tumor cell membranes, bind anti-Gal and activate complement. The complement cleavage peptides C5a and C3a recruit inflammatory cells and APC into the treated lesion. Anti-Gal further opsonizes the tumor cells and targets them for effective uptake by recruited APC, via Fcγ receptors. These APC transport internalized tumor cells to draining lymph nodes, and present immunogenic tumor antigen peptides for activation of tumor specific T cells. The present study demonstrates the ability of α-gal glycolipids treatment to prevent development of metastases at distant sites and to protect against tumor challenge in the treated mice. Adoptive transfer studies indicate that this protective immune response is mediated by CD8+ T cells, activated by tumor lesions turned vaccine. This T cell activation is potent enough to overcome the suppressive activity of Treg cells present in tumor bearing mice, however it does not elicit an autoimmune response against antigens on normal cells. Insertion of α-gal glycolipids and subsequent binding of anti-Gal are further demonstrated with human melanoma cells, suggesting that intratumoral injection of α-gal glycolipids is likely to elicit a protective immune response against micrometastases also in cancer patients.