Structure-function analysis and therapeutic efficacy of antibodies to fungal melanin for melanoma radioimmunotherapy.

Metastatic melanoma remains difficult to treat despite recent approvals of several new drugs. Recently we reported encouraging results of Phase I clinical trial of radiolabeled with 188Re murine monoclonal IgM 6D2 to melanin in patients with Stage III/IV melanoma. Subsequently we generated a novel murine IgG 8C3 to melanin. IgGs are more amenable to humanization and cGMP (current Good Manufacturing Practice) manufacturing than IgMs. We performed comparative structural analysis of melanin-binding IgM 6D2 and IgG 8C3. The therapeutic efficacy of 213Bi- and 188Re-labeled 8C3 and its comparison with anti-CTLA4 immunotherapy was performed in B16-F10 murine melanoma model. The primary structures of these antibodies revealed significant homology, with the CDRs containing a high percentage of positively charged amino acids. The 8C3 model has a negatively charged binding surface and significant number of aromatic residues in its H3 domain, suggesting that hydrophobic interactions contribute to the antibody-melanin interaction. Radiolabeled IgG 8C3 showed significant therapeutic efficacy in murine melanoma, safety towards healthy melanin-containing tissues and favorable comparison with the anti-CTLA4 antibody. We have demonstrated that antibody binding to melanin relies on both charge and hydrophobic interactions while the in vivo data supports further development of 8C3 IgG as radioimmunotherapy reagent for metastatic melanoma.

Safety and efficacy of 188-rhenium-labeled antibody to melanin in patients with metastatic melanoma.

There is a need for effective "broad spectrum" therapies for metastatic melanoma which would be suitable for all patients. The objectives of Phase Ia/Ib studies were to evaluate the safety, pharmacokinetics, dosimetry, and antitumor activity of (188)Re-6D2, a 188-Rhenium-labeled antibody to melanin. Stage IIIC/IV metastatic melanoma (MM) patients who failed standard therapies were enrolled in both studies. In Phase Ia, 10 mCi (188)Re-6D2 were given while unlabeled antibody preload was escalated. In Phase Ib, the dose of (188)Re-6D2 was escalated to 54 mCi. SPECT/CT revealed (188)Re-6D2 uptake in melanoma metastases. The mean effective half-life of (188)Re-6D2 was 12.4 h. Transient HAMA was observed in 9 patients. Six patients met the RECIST criteria for stable disease at 6 weeks. Two patients had durable disease stabilization for 14 weeks and one for 22 weeks. Median overall survival was 13 months with no dose-limiting toxicities. The data demonstrate that (188)Re-6D2 was well tolerated, localized in melanoma metastases, and had antitumor activity, thus warranting its further investigation in patients with metastatic melanoma.

Pre-clinical evaluation and efficacy studies of a melanin-binding IgM antibody labeled with 188Re against experimental human metastatic melanoma in nude mice.

PURPOSE: Currently there is no satisfactory treatment for metastatic melanoma. Radioimmunotherapy (RIT) uses the antigen-antibody interaction to deliver lethal radiation to target cells. Recently we established the feasibility of targeting melanin in tumors with 188-Rhenium ((188)Re)-labeled 6D2 mAb to melanin. Here we carried out pre-clinical development of (188)Re-6D2 to accrue information necessary for a Phase I trial in patients with metastatic melanoma. RESULTS: TCEP proved to be effective in generating a sufficient number of -SH groups on 6D2 to ensure high radiolabeling yields with (188)Re and preserved its structural integrity. (188)Re-6D2 was quickly cleared from the blood with the half-life of approximately 5 hrs and from the body--with the half-life of 10 hr. The doses of 0.5, 1.0 and 1.5 mCi significantly (p < 0.05) slowed down A2058 tumor growth in nude mice, also causing release of melanin into the extracellular space which could provide additional target for repeated treatments. Transient effects of RIT on WBC and platelet counts resolved by Day 14 post-treatment. EXPERIMENTAL DESIGN: Tris(2-Carboxyethyl) Phosphine Hydrochloride (TCEP) was evaluated as potential agent for generation of -SH groups on 6D2 mAb. TCEP-treated 6D2 mAb was radiolabeled with (188)Re and its radiochemical purity and stability was measured by ITLC and HPLC and its immunoreactivity--by melanin-binding ELISA. The pharmacokinetics, therapeutic efficacy and acute hematologic toxicity studies were performed in nude mice bearing lightly pigmented A2058 human metastatic melanoma tumors. CONCLUSIONS: We have developed radiolabeling and quality control procedures for melanin-binding (188)Re-6D2 mAb which made possible currently an on-going Phase I clinical trial in patients with metastatic melanoma.

Regulation of leukocyte-endothelium interaction and leukocyte transendothelial migration by intercellular adhesion molecule 1-fibrinogen recognition.

Although primarily recognized for its role in hemostasis, fibrinogen is also required for competent inflammatory reactions in vivo. It is now shown that fibrinogen promotes adhesion to and migration across an endothelial monolayer of terminally differentiated myelomonocytic cells. This process does not require chemotactic/haptotactic gradients or cytokine stimulation of the endothelium and is specific for the association of fibrinogen with intercellular adhesion molecule 1 (ICAM-1) on endothelium. Among other adhesive plasma proteins, fibronectin fails to increase the binding of leukocytes to endothelium, or transendothelial migration, whereas vitronectin promotes the binding but not the migration. The fibrinogen-mediated leukocyte adhesion and transendothelial migration could be inhibited by a peptide from the fibrinogen gamma-chain sequence N117NQKIVNL-KEKVAQLEA133, which blocks the binding of fibrinogen to ICAM-1. This interaction could also be inhibited by new anti-ICAM-1 monoclonal antibodies that did not affect the ICAM-1-CD11a/CD18 recognition, thus suggesting that the fibrinogen binding site on ICAM-1 may be structurally distinct from regions previously implicated in leukocyte-endothelium interaction. Therefore, binding of fibrinogen to vascular cell receptors is sufficient to initiate (i) increased leukocyte adhesion to endothelium and (ii) leukocyte transendothelial migration. These two processes are the earliest events of immune inflammatory responses and may also contribute to atherosclerosis.

Structural recognition of a novel fibrinogen gamma chain sequence (117-133) by intercellular adhesion molecule-1 mediates leukocyte-endothelium interaction.

In addition to its role in hemostasis, fibrinogen is obligatorily required to mount competent inflammatory responses in vivo. A molecular prerequisite of fibrinogen-dependent inflammation may reside in its ability to associate with intercellular adhesion molecule-1 (ICAM-1), and enhance monocyte adhesion to endothelium by bridging the two cell types. Structure-function characterization of the novel ICAM-1 recognition of fibrinogen was carried out by synthetic peptidyl mimicry of the fibrinogen gamma chain. A novel peptide sequence, N117NQ-KIVNLKEKVAQLEA133, designated gamma 3, dose-dependently inhibited (IC50 approximately 20-40 micrograms/ml) binding of 125I-fibrinogen to endothelial cells or ICAM-1-expressing B lymphoblastoid Daudi cells. In contrast, none of the previously identified vascular cell fibrinogen interacting sequences was effective. Increasing concentrations of gamma 3 completely inhibited fibrinogen-mediated adhesion of peripheral blood mononuclear cells or vitamin D3-differentiated monocytic HL-60 cells to endothelium, but did not affect leukocyte-endothelium interaction in the absence of fibrinogen. 125I-Labeled gamma 3 bound specifically and saturably to genetically engineered ICAM-1 transfectants, but not to control non-transfected cells, and associated with ICAM-1 on cytokine-activated endothelium with a Kd of 34 microM. Consistent with functional recognition of ICAM-1, immobilized gamma 3 supported adhesion of JY lymphoblasts in a dose-dependent reaction inhibited by monoclonal antibodies to ICAM-1. We conclude that a novel fibrinogen gamma 3 sequence N117NQKIVNLKEKVAQLEA133 binds to ICAM-1 and modulates ICAM-1-dependent adhesion. These findings define the structural basis of fibrinogen:ICAM-1 recognition and provide a potential selective target for inhibiting fibrinogen-dependent inflammatory responses.

Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody.

The ability of antibodies to neutralize diverse primary isolates of human immunodeficiency virus-type 1 in vitro has been questioned, with implications for the likely efficacy of vaccines. A recombinant human antibody to envelope glycoprotein gp120 was generated and used to show that primary isolates are not refractory to antibody neutralization. The recombinant antibody neutralized more than 75 percent of the primary isolates tested at concentrations that could be achieved by passive immunization, for example, to interrupt maternal-fetal transmission of virus. The broad specificity and efficacy of the antibody implies the conservation of a structural feature on gp120, which could be important in vaccine design.

Use of anti-peptide antibodies for the design of antigen-specific immune complex assays.

A simple and sensitive method is described for the detection of circulating immune complexes (ICs) in an antigen-specific manner. The method is based on the use of anti-peptide antibodies as solid-phase capture reagents to bind antigen which is complexed to serum antibodies. The bound serum antibody is detected with a labelled second antibody. The method requires that the anti-peptide antibodies bind native protein efficiently, and that the anti-peptide antibodies do not compete with antibodies raised against the native protein which are involved in IC formation. Two anti-peptide antibodies specific for the hepatitis B surface antigen (HBsAg) and the hepatitis B e antigen (HBeAg), which possessed the requisite characteristics, were chosen as models for IC assay development. The solid-phase, anti-peptide based assays efficiently detected HBsAg and HBeAg-containing ICs in preformed antigen/antibody mixtures and in the serum of chronically infected hepatitis B patients.

Production of infectious hepatitis delta virus in vitro and neutralization with antibodies directed against hepatitis B virus pre-S antigens.

Hepatitis delta virus (HDV) particles were produced in Huh7 human hepatoma cells by transfection with cloned hepatitis B virus (HBV) DNA and HDV cDNA. The particles were characterized by their buoyant density, the presence of encapsidated viral RNA, and their ability to infect primary cultures of chimpanzee hepatocytes. Successful infection was evidenced by the appearance of increasing amounts of intracellular HDV RNA after exposure to particles. Infection was prevented when particles were incubated with antibodies directed against synthetic peptides specific for epitopes of the pre-S1 or pre-S2 domains of the HBV envelope proteins before exposure to hepatocytes. These data demonstrate that HDV particles produced in vitro are infectious and indicate (i) that infectious particles are coated with HBV envelope proteins that contain the pre-S1 and pre-S2 regions, (ii) that epitopes of the pre-S1 and pre-S2 domains of HBV envelope proteins are exposed at the surface of HDV particles, and (iii) that antibodies directed against those epitopes have neutralizing activity against HDV.

Immunization of hu-PBL-SCID mice and the rescue of human monoclonal Fab fragments through combinatorial libraries.

Antibodies are usually prepared from recently boosted animals and reflect ongoing immune responses. In humans, this is restrictive as ethical constraints generally prevent antigen-boosting. Therefore the rich memory compartment of human antibody responses remains largely untapped. Severe combined immune deficiency (SCID) mice populated with human cells allow the stimulation of human antibody memory without the usual constraints. Here we show how peripheral blood lymphocytes can be stimulated by antigen to produce large secondary responses after transfer to SCID mice. Specific monoclonal human Fab fragments can then be isolated from the mice by repertoire cloning even when the human donor's last contact with antigen was more than 17 years ago.

Autoantibody production in hepatitis B e antigen transgenic mice elicited with a self T-cell peptide and inhibited with nonself peptides.

Studies in hepatitis B e antigen (HBeAg)-expressing transgenic mice indicate that self tolerance to two T-cell determinants on the same transgenic self molecule can differ markedly. The dominant T-cell site on HBeAg is tolerogenic, whereas a proportion of T cells recognizing a second T-cell site evade tolerance induction, persist in the periphery, and can be activated in vivo by a single injection of a 12-residue T-cell self peptide. The self-reactive T cells mediate in vivo autoantibody production sufficient to neutralize detection of the autoantigen in serum. Furthermore, autoantibody production can be inhibited by nonself peptides that compete with the self peptide for binding to major histocompatibility complex molecules. This model illustrates that T cells specific for an immunogenic T-cell site on a nonsequestered autoantigen can escape tolerance induction and, more importantly, can mediate autoreactivity in vivo. Furthermore, these results suggest that synthetic T-cell sites may be useful as immunotherapeutic agents for the purpose of circumventing nonresponse to HBeAg during persistent hepatitis B virus infection.

Importance of subtype in the immune response to the pre-S(2) region of the hepatitis B surface antigen. I. T cell fine specificity.

Previous studies of murine T cell recognition of the pre-S(2) region of the hepatitis B surface Ag (HBsAg) identified high (H-2b,d,q), intermediate (H-2s,k), and low to nonresponder (H-2f) haplotypes. However, these studies utilized the y subtype of HBsAg. The purpose of this study was to examine the influence of viral subtype on T cell recognition of the pre-S(2) region and to identify specific T cell recognition sites in a panel of H-2 congenic strains. Immunization with pre-S(2) containing HBsAg particles of the d and y subtypes indicated that T cell recognition of the pre-S(2) region is predominantly subtype-specific in murine strains of eight different H-2 haplotypes. Furthermore, the B10.M strain (H-2f) classified as a T cell nonresponder to the y subtype of the pre-S(2) region responds efficiently to the d subtype, indicating that pre-S(2) responder status can be subtype-dependent as well as subtype-specific. Studies using a truncated pre-S(2) polypeptide and synthetic peptides illustrated that the C-terminal sequence (p148-174) of the pre-S(2) region is the dominant focus of T cell recognition in multiple murine strains. Specifically, 17 distinct T cell recognition sites were defined within the C-terminal half of the pre-S(2) region. The fine specificity of T cell recognition of the pre-S(2) region was dependent on the H-2 haplotype of the responding strain. T cell recognition of all 17 sites was subtype specific, which is consistent with the fact that the C-terminal sequence is highly polymorphic between the d and y subtypes of the pre-S(2) region. Lastly, it was shown that the ability of synthetic peptides to elicit T cells cross-reactive with the native pre-S(2) region was variable and depended on the nature of the immunizing peptide. The pre-S(2)-containing HBsAg vaccines currently in clinical trials are composed of ra single subtype, either d or y. The results of this study suggest that both subtypes should be incorporated to increase the frequency of T cell responders to the pre-S(2) region, and to insure Th cell memory relevant to infection with hepatitis B virus of either the d or y subtypes.

Distinction between immunogenicity and tolerogenicity among HBcAg T cell determinants. Influence of peptide-MHC interaction.

One purpose of this study was to examine the concept of T cell immunodominance employing a neonatal tolerance model. The extent to which a single T cell recognition site can represent the total T cell response to hepatitis B core Ag (HBcAg) was examined in the B10.S and B10 murine strains. It was shown that the entire B10.S T cell response to HBcAg was focused on a single immunodominant site represented by residues 120-131. This was demonstrated by exposing B10.S neonatal mice to p120-140 or p120-131, which resulted in a state of T cell tolerance to the entire HBcAg. In contrast, p120-140 contained an immunogenic T cell site for B10 mice, p129-140, but this site was nontolerogenic. Similarly, injection of p120-140 into (B10.S X B10)F1 neonatal mice resulted in tolerization of p120-131-specific, I-As-restricted T cells, but not of p129-140-specific, I-Ab-restricted T cells. The second purpose of this study was to attempt to explain the immunologic basis of an immunogenic yet nontolerogenic T cell determinant. It was shown that the p120-131 T cell site, which is immunogenic and tolerogenic in B10.S mice, could be converted into an immunogenic/nontolerogenic T cell site by a single amino acid substitution in either residue 127 or 129. Residues 127 and 129 were previously shown to be involved in interaction with MHC class II molecules (agretopic). These results demonstrated that the relative avidity of a peptide-MHC interaction can influence T cell tolerance induction. Furthermore, the results suggest that a higher threshold of peptide-MHC avidity may be required to induce T cell tolerance as compared to the threshold of peptide-MHC avidity required to immunize T cells.

Comparative immunogenicity of hepatitis B virus core and E antigens.

The nucleocapsid (hepatitis B core Ag (HBcAg] of the hepatitis B virus is a particulate Ag composed of a single polypeptide (p21). Although a non-particulate form of HBcAg designated hepatitis B e Ag (HBeAg) shares significant amino acid identity, the immune responses to these Ag appear to be regulated independently. This report describes the use of recombinant HBcAg and HBeAg to examine and compare murine T cell and B cell recognition of these related Ag. The HBcAg preparation was stable at pH 7.2 and 9.6 and expressed HBc antigenicity. However, the antigenicity of the HBeAg preparation was pH dependent. At pH 9.6 the HBeAg preparation was non-particulate and expressed HBe antigenicity exclusively; however, at pH 7.2 it was particulate and expressed both HBc and HBe antigenicities. Although this "hybrid" particle most likely does not exist naturally, it is a unique research reagent to investigate the interrelationship between HBcAg and HBeAg. HBcAg was significantly more immunogenic in terms of in vivo antibody production as compared to either the non-particulate or particulate forms of HBeAg. Nevertheless, in most murine strains HBcAg and HBeAg were equivalently immunogenic and crossreactive at the level of T cell activation. The disparity between anti-HBc and anti-HBe antibody production is best explained by the observation that HBcAg can function as a T cell-independent Ag whereas HBeAg is T cell dependent even when present within the same particulate structure as HBcAg. Furthermore, HBcAg was shown to function efficiently as an immunologic carrier moiety for the DNP hapten in athymic as well as euthymic mice in contrast to conventional carrier proteins. These results have implications relevant to the human immune responses to HBcAg and HBeAg during infection, and to vaccine development.

Hepatitis B synthetic immunogen comprised of nucleocapsid T-cell sites and an envelope B-cell epitope.

Previous studies located T-cell recognition of the nucleocapsid of the hepatitis B virus (HBcAg) to residues 120-140 in mice bearing the H-2s or H-2b haplotypes. Herein, we demonstrate that B10.S (H-2s) and B10 (H-2b) H-2 congenic strains recognize distinct T-cell sites within the p120-140 (a synthetic peptide corresponding to residues 120-140 of HBcAg) sequence defined by p120-131 and p129-140, respectively. Peptide p120-131 stimulates B10.S HBcAg-primed T cells, and reciprocally p120-131-primed T cells recognize HBcAg. Similarly, the p129-140 sequence is a T-cell recognition site relevant to the native HBcAg in the B10 strain. It is also shown that these 12-residue peptides efficiently prime T-helper cells, which are capable of eliciting antibody production to HBcAg in vivo. These observations prompted us to examine the ability of the HBcAg-specific p120-140 sequence to function as a T-cell carrier moiety as a component of a totally synthetic hepatitis B vaccine. For this purpose a synthetic B-cell epitope from the pre-S(2) region (p133-140) of the viral envelope was chosen because this sequence represents a dominant antibody-binding site of the envelope. Immunization of B10.S and B10 strains with the synthetic composite peptide c120-140-(133-140) elicited anti-peptide antibody production, which was crossreactive with the native viral envelope. Furthermore, c120-140-(133-140) immunization primed p120-131-specific T cells in the B10.S strain and p129-140-specific T cells in the B10 strain, which recognized HBcAg and provided T-helper cell function for anti-envelope antibody production in vivo. These results demonstrate the feasibility of constructing complex synthetic immunogens that represent multiple proteins of a pathogen and are capable of engaging both T and B cells relevant to the native antigens.

Antibody production to the nucleocapsid and envelope of the hepatitis B virus primed by a single synthetic T cell site.

The nucleocapsid (HBcAg) of the hepatitis B virus (HBV) can induce antibody responses via both a T-cell dependent and a T-cell independent pathway and is highly immunogenic during infection. We have examined the T-cell determinants of the antigen and find that HBcAg-specific helper T cells (TH) can help B cells produce antibody against envelope (HBsAg) antigens as well as HBcAg, even though these antigens are found on separate molecules. We have also been able to prime helper T cells with synthetic T-cell epitopes of HBcAg; helper cells primed with a single synthetic epitope can induce B cells to produce antibody that reacts with multiple HBsAg epitopes. One problem with the development of an HBV vaccine is that some vaccinees and patients do not respond to HBsAg directly; our results indicate that this problem can be circumvented using the response to HBcAg.

Immune response to hepatitis B virus core antigen (HBcAg): localization of T cell recognition sites within HBcAg/HBeAg.

Hepatitis B virus nucleocapsid particles (HBcAg) can function as a T cell-independent antigen when injected into athymic mice. However, immunization of euthymic mice with HBcAg results in dramatically increased anti-HBc titers. Therefore we have examined the murine T cell response to HBcAg in terms of immunogenicity, the influence of H-2-linked genes, and the fine specificity of T cell recognition using synthetic peptide analogs. The HBcAg was shown to be an extremely efficient immunogen in terms of T cell activation as measured by the in vivo dose required to induce T cell sensitization (1.0 microgram), and the minimal in vitro concentration required to elicit interleukin 2 (IL 2) production (0.03 ng/ml). The degree of T cell immunogenicity of HBcAg and its ability to directly activate B cells most likely explain the enhanced humoral response to HBcAg in euthymic mice and HBV-infected patients. The influence of H-2-linked genes on the humoral response to HBcAg was discernable, and high responder (H-2k,s,d), intermediate responder (H-2b,f), and low responder (H-2p) haplotypes were identified. The H-2-linked regulation of the T cell response correlated with in vivo anti-HBc production. Examination of the fine specificity of T cell recognition revealed HBcAg-specific T cells from a variety of strains recognize multiple but distinct sites within the HBcAg/HBeAg sequence. T cell recognition sites were defined by small (16 to 21 residue) synthetic peptides. Each strain recognized a predominant T cell determinant, and the fine specificity of this recognition process was dependent on the H-2 haplotype of the responding strain. For example H-2s,b strains recognized p120-140, H-2f,q strains recognized p100-120, and H-2d mice recognized p85-100 predominantly. Because these sequences are common to both HBcAg and a nonparticulate form of the antigen termed HBeAg, these results indicate that HBcAg and HBeAg are highly cross-reactive at the T cell level although they are serologically distinct. These findings may have clinical relevance, because T cell sensitization to HBeAg and the subsequent seroconversion to anti-HBe status correlates with viral clearance during hepatitis B infection.

A single 10-residue pre-S(1) peptide can prime T cell help for antibody production to multiple epitopes within the pre-S(1), pre-S(2), and S regions of HBsAg.

The purpose of this study was to identify and characterize T cell and B cell recognition sites within the pre-S(1) region of HBsAg/p43, and to then analyze functional T cell-B cell interactions at the level of in vivo antibody production. The results indicate: three peptide sequences within the pre-S(1) region of HBsAg were identified which can induce and elicit HBsAg/p43-specific T cell proliferation; a 10-amino acid peptide, p12-21, defines one pre-S(1)-specific T cell recognition site, and residues 18 and 19 are critical to the recognition process; the p12-21 sequence can function as a T cell carrier for a synthetic B cell epitope within the pre-S(2) region; the p94-117 sequence contains at least two T cell recognition sites; five distinct, pre-S(1)-specific antibody binding sites were identified; synthetic pre-S(1) region T cell determinants can prime in vivo antibody production to multiple B cell epitopes within the pre-S(2) and S regions, as well as within the pre-S(1) region; the specificity of the primed T cell population can influence the specificity of the B cell response; and T cell recognition of pre-S(1) region peptides is regulated by H-2-linked genes.

T-cell recognition of pre-S regions of HBsAg can bypass nonresponse to the S region.

The objective of the studies reported herein was to identify and characterize T cell and B cell recognition sites within the pre-S regions of HBsAg/p39, and to analyze functional T-cell-B cell interactions at the level of in vivo antibody production. The results indicate: (1) several peptides within the pre-S(1) region of HBsAg were identified which can induce and elicit HBsAg/p39-specific T-cell proliferation; (2) a 10 amino acid peptide, p12-21, and the 94-117 sequence define pre-S(1)-specific T-cell recognition sites; (3) five distinct, pre-S(1)-specific antibody binding sites and 2 pre-S(2)-specific antibody binding sites were identified; (4) synthetic pre-S(1) region T-cell determinants can prime in vivo antibody production to multiple B-cell epitopes within the pre-S(2) and S regions, as well as within the pre-S(1) region; and (5) specificity of the primed T cell population can influence the specificity of the B-cell response.