Metal-triggered conformational reorientation of a self-peptide bound to a disease-associated HLA-B*27 subtype.

Conformational changes of major histocompatibility complex (MHC) antigens have the potential to be recognized by T cells and may arise from polymorphic variation of the MHC molecule, the binding of modifying ligands, or both. Here, we investigated whether metal ions could affect allele-dependent structural variation of the two minimally distinct human leukocyte antigen (HLA)-B*27:05 and HLA-B*27:09 subtypes, which exhibit differential association with the rheumatic disease ankylosing spondylitis (AS). We employed NMR spectroscopy and X-ray crystallography coupled with ensemble refinement to study the AS-associated HLA-B*27:05 subtype and the AS-nonassociated HLA-B* 27:09 in complex with the self-peptide pVIPR (RRKWRRWHL). Both techniques revealed that pVIPR exhibits a higher degree of flexibility when complexed with HLA-B*27:05 than with HLA-B*27:09. Furthermore, we found that the binding of the metal ion Cu2+ or Ni2+, but not Mn2+, Zn2+, or Hg2+, affects the structure of a pVIPR-bound HLA-B*27 molecule in a subtype-dependent manner. In HLA-B*27:05, the metals triggered conformational reorientations of pVIPR, but no such structural changes were observed in the HLA-B*27:09 subtype, with or without bound metal ion. These observations provide the first demonstration that not only major histocompatibility complex class II, but also class I, molecules can undergo metal ion-induced conformational alterations. Our findings suggest that metals may have a role in triggering rheumatic diseases such as AS and also have implications for the molecular basis of metal-induced hypersensitivities and allergies.

Targeting Tumor Associated Phosphatidylserine with New Zinc Dipicolylamine-Based Drug Conjugates.

A series of zinc(II) dipicolylamine (ZnDPA)-based drug conjugates have been synthesized to probe the potential of phosphatidylserine (PS) as a new antigen for small molecule drug conjugate (SMDC) development. Using in vitro cytotoxicity and plasma stability studies, PS-binding assay, in vivo pharmacokinetic studies, and maximum tolerated dose profiles, we provided a roadmap and the key parameters required for the development of the ZnDPA based drug conjugate. In particular, conjugate 24 induced tumor regression in the COLO 205 xenograft model and exhibited a more potent antitumor effect with a 70% reduction of cytotoxic payload compared to that of the marketed irinotecan when dosed at the same regimen. In addition to the validation of PS as an effective pharmacodelivery target for SMDC, our work also provided the foundation that, if applicable, a variety of therapeutic agents could be conjugated in the same manner to treat other PS-associated diseases.

A clinical trial to evaluate the safety and immunogenicity of the LEISH-F1+MPL-SE vaccine when used in combination with meglumine antimoniate for the treatment of cutaneous leishmaniasis.

Forty-four adult patients with cutaneous leishmaniasis (CL) were enrolled in a randomized, double-blind, controlled, dose-escalating clinical trial and were randomly assigned to receive three injections of either the LEISH-F1+MPL-SE vaccine (consisting of 5, 10, or 20 µg recombinant Leishmania polyprotein LEISH-F1 antigen+25 µg MPL-SE adjuvant) (n=27), adjuvant alone (n=8), or saline placebo (n=9). The study injections were given subcutaneously on Days 0, 28, and 56, and the patients were followed through Day 336 for safety, immunological, and clinical evolution endpoints. All patients received chemotherapy with meglumine antimoniate starting on Day 0. The vaccine was safe and well tolerated. Nearly all vaccine recipients and no adjuvant-alone or placebo recipients demonstrated an IgG antibody response to LEISH-F1 at Day 84. Also at Day 84, 80% of vaccine recipients were clinically cured, compared to 50% and 38% of adjuvant-alone and placebo recipients. The LEISH-F1+MPL-SE vaccine was safe and immunogenic in CL patients and appeared to shorten their time to cure when used in combination with meglumine antimoniate chemotherapy.

Rates and equilibria for probe capture by an antibody with infinite affinity.

Probe-capture systems based on proteins and synthetic ligands have become important for new analytical and imaging applications. We have used kinetic measurements of luminescence and measurements of binding by isothermal calorimetry to determine essential rate and equilibrium constants for a system that permanently captures modified DOTA chelates for positron imaging. We used that information along with previous results to quantitatively characterize the behavior of this system in vitro and in vivo. Under physiological conditions at 37 degrees C, the equilibrium dissociation constant for yttrium S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetate from antibody 2D12.5 is 2.0 (+/- 0.4) x 10(-9) M and the dissociation rate constant is 7.0 (+/- 0.7) x 10(-3) s(-1), leading to an inferred association rate constant of 3.5 x 10(6) M(-1) s(-1). Using these values to interpret data from earlier experiments leads to the rate constant 2.5 x 10(-2) s(-1) for covalent attachment of bound yttrium S-2-(4-acrylamidobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetate to the G54C mutant of antibody 2D12.5. These values lead to a model for the detailed behavior of the latter system for tumor imaging in vivo that is consistent with experimental observations.

[Gd@C(82)(OH)(22)](n) nanoparticles induce dendritic cell maturation and activate Th1 immune responses.

Dendritic cells play a pivotal role in host immune defense, such as elimination of foreign pathogen and inhibition of tumorigenesis. In this paper, we report that [Gd@C(82)(OH)(22)](n) could induce phenotypic maturation of dendritic cells by stimulating DC production of cytokines including IL-12p70, upregulating DC co-stimulatory (CD80, CD83, and CD86) and MHC (HLA-A,B,C and HLA-DR) molecules, and switching DCs from a CCL5-responsive to a CCL19-responsive phenotype. We found that [Gd@C(82)(OH)(22)](n) can induce dendritic cells to become functionally mature as illustrated by their capacity to activate allogeneic T cells. Mice immunized with ovalbumin in the presence of [Gd@C(82)(OH)(22)](n) exhibit enhanced ovalbumin-specific Th1-polarized immune response as evidenced by the predominantly increased production of IFNgamma, IL-1beta, and IL-2. The [Gd@C(82)(OH)(22)](n) nanoparticle is a potent activator of dendritic cells and Th1 immune responses. These new findings also provide a rational understanding of the potent anticancer activities of [Gd@C(82)(OH)(22)](n) nanoparticles reported previously.

Radioimmunotherapy of experimental human metastatic melanoma with melanin-binding antibodies and in combination with dacarbazine.

PURPOSE: Melanin has emerged as an attractive target for radioimmunotherapy (RIT) of melanoma, and a radiolabeled monoclonal antibody (mAb) 6D2 to melanin is currently in clinical evaluation. We investigated two approaches to improve the targeting of radiation to tumors using melanin-binding mAbs: (a) the use of an additional mAb to melanin could provide information on whether using antibodies to melanin can serve as a general approach to development of therapeutics for melanoma, and (b) as melanin targeting involves the antibody binding to extracellular melanin released from necrotic melanoma cells, we hypothesized that the administration of a chemotherapeutic agent followed by RIT would facilitate the delivery of radiation to the tumors due to the increased presence of free melanin. EXPERIMENTAL DESIGN: We evaluated the therapeutic efficacy of two melanin-binding IgM mAbs labeled with (188)Re (6D2 and 11B11). We compared the efficacy of RIT with (188)Re-6D2 to chemotherapy with dacarbazine (DTIC) and to combined chemotherapy and RIT in human metastatic melanoma-bearing nude mice. RESULTS: Therapeutic efficacy of (188)Re-labeled 6D2 and 11B11 was comparable despite differences in their affinity and binding site numbers. Comparison of chemotherapy with DTIC and RIT revealed that RIT was more effective in slowing tumor growth in mice. Administration of DTIC followed by RIT was more effective than either modality alone. CONCLUSIONS: These results provide encouragement for the development of RIT for melanoma with melanin-binding mAbs and suggest that combining chemotherapy and RIT may be a promising approach for the treatment of metastatic melanoma.

Synthetic and immunological studies of 5'-N-phenylacetyl sTn to develop carbohydrate-based cancer vaccines and to explore the impacts of linkage between carbohydrate antigens and carrier proteins.

5'- N-Phenylacetyl sTn (sTnNPhAc), an unnatural derivative of sTn antigen expressed by many tumors, and its alpha-linked protein conjugates were prepared and investigated to explore glycoconjugate cancer vaccines. sTnNPhAcalpha-KLH elicited a robust T cell dependent immunity. The antiserum derived from sTnNPhAcalpha- or sTnNPhAcbeta-KLH-inoculated mice was similarly reactive to sTnNPhAcalpha and sTnNPhAcbeta but showed very little reactivity to sTn, NeuNPhAcalpha(2,3)GalNAc--a regioisomer of sTnNPhAc, isolated phenylacetyl group, and the linker employed to conjugate sTnNPhAc and carrier protein. It was concluded that the sTnNPhAc-elicited immunity was specific for the whole antigen rather than the phenylacetyl group or other partial structures of sTnNPhAc and that the reducing end configuration or linkage of sTnNPhAc did not affect its immunological identity. It was also concluded that a new linker designed to conjugate carbohydrates and proteins did not provoke any immune reaction and that the linker, as well as the associated new and convenient coupling strategy, can be safely used for the development of glycoconjugate vaccines.

Attomole detection of hemagglutinin molecule of influenza virus by combining an electrochemiluminescence sensor with an immunoliposome that encapsulates a Ru complex.

An immunoliposome (80 nm in diameter) encapsulating a Ru complex with two aminobutyl moieties was prepared to detect the presence of hemagglutinin molecules, which play an important role in influenza virus infection. The highly sensitive detection was accomplished by electrochemiluminescence (ECL) from the Ru complex adsorbed onto Au electrodes after competitive immunoreactions. This method clarified that the adsorption of the Ru complex onto the electrode was an important factor in obtaining high sensitivity. Optimization of the analytical conditions enabled determination of the hemagglutinin molecules of the influenza virus in the concentration range of 3 x 10(-14) (6 x 10(-19) mol/50 microL sample) to 2 x 10(-12) g/mL. The sensitivity was far superior to that obtained by conventional ELISA as well as to that obtained by biosensors and reported thus far.

PET (positron emission tomography) imaging of biomolecules using metal-DOTA complexes: a new collaborative challenge by chemists, biologists, and physicians for future diagnostics and exploration of in vivo dynamics.

Recently, PET has been paid a great deal of attention as a non-invasive imaging method. In this review, the recent advances of PET using biomolecules, such as peptides, monoclonal antibodies, proteins, oligonucleotides, and glycoproteins will be described. So far, PET of biomolecules has been mainly used for diagnosis of cancers. The biomolecules have been conjugated with the DOTA ligand, labeled with radiometals as the beta+ emitter, and targeted to specific tumors, where they have enabled visualization of even small metastatic lesions, due to the high sensitivity of the PET scanners. Some of the biomolecules have been used not only for PET diagnosis, but also for radiotherapeutic treatments by simply changing the radiometals to beta(-) emitters. Collaborative work between chemists, biologists, and physicians will be important for the future of biomolecule-based targeting and diagnosis.

In vitro and in vivo characterization of 64Cu-labeled Abegrin, a humanized monoclonal antibody against integrin alpha v beta 3.

Abegrin (MEDI-522 or Vitaxin), a humanized monoclonal antibody against human integrin alpha(v)beta(3), is in clinical trials for cancer therapy. In vivo imaging using Abegrin-based probes is needed for better treatment monitoring and dose optimization. Here, we conjugated Abegrin with macrocyclic chelating agent 1,4,7,10-tetra-azacylododecane N,N',N'',N'''-tetraacetic (DOTA) at five different DOTA/Abegrin ratios. The conjugates were labeled with (64)Cu (half-life = 12.7 hours) and tested in three human (U87MG, MDA-MB-435, and PC-3) and one mouse (GL-26) tumor models. The in vitro and in vivo effects of these (64)Cu-DOTA-Abegrin conjugates were evaluated. The number of DOTA per Abegrin varied from 1.65 +/- 0.32 to 38.53 +/- 5.71 and the radiolabeling yield varied from 5.20 +/- 3.16% to 88.12 +/- 6.98% (based on 2 mCi (64)Cu per 50 microg DOTA-Abegrin conjugate). No significant difference in radioimmunoreactivity was found among these conjugates (between 59.78 +/- 1.33 % and 71.13 +/- 2.58 %). Micro-positron emission tomography studies revealed that (64)Cu-DOTA-Abegrin (1,000:1) had the highest tumor activity accumulation (49.41 +/- 4.54% injected dose/g at 71-hour postinjection for U87MG tumor). The receptor specificity of (64)Cu-DOTA-Abegrin was confirmed by effective blocking of MDA-MB-435 tumor uptake with coadministration of nonradioactive Abegrin. (64)Cu-DOTA-IgG exhibited background level tumor uptake at all time points examined. Integrin alpha(v)beta(3)-specific tumor imaging using (64)Cu-DOTA-Abegrin may be translated into the clinic to characterize the pharmacokinetics, tumor targeting efficacy, dose optimization, and dose interval of Abegrin and/or Abegrin conjugates. Chemotherapeutics or radiotherapeutics using Abegrin as the delivering vehicle may also be effective in treating integrin alpha(v)beta(3)-positive tumors.

Immunological control of methicillin-resistant Staphylococcus aureus (MRSA) infection in an immunodeficient murine model of thermal injuries.

Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), is a major cause of sepsis in patients who are immunosuppressed by their burns. In this study, an immunological regulation of MRSA infection was attempted in a mouse model of thermal injury. SCIDbg mice were resistant to MRSA infection, while SCIDbgMN mice (SCIDbg mice depleted of neutrophils and macrophages (Mphi)) were susceptible to the same infection. Also, thermally injured SCIDbg mice were shown to be susceptible to MRSA infection. On the other hand, the resistance of SCIDbgMN mice to the infection was completely recovered after an inoculation with Mphi from normal mice. However, anti-MRSA resistance was not shown in SCIDbgMN mice inoculated with Mphi from thermally injured mice. Mphi from MRSA-infected thermally injured mice were identified as alternatively activated Mphi, and Mphi from MRSA-infected unburned mice were characterized as classically activated Mphi. Mphi from thermally injured SCIDbg mice previously treated with 2-carboxyethylgermanium sesquioxide (Ge-132) protected SCIDbgMN mice against MRSA infection. Ge-132 has been described as an inhibitor of alternatively activated Mphi generation. These results suggest that MRSA infection in thermally injured patients is controlled immunologically through the induction of anti-MRSA effector cells and elimination of burn-associated alternatively activated Mphi, which are cells that inhibit the generation of classically activated Mphi.

Neonatal tolerance to a Th2-mediated autoimmune disease generates CD8+ Tc1 regulatory cells.

Immunological tolerance can be achieved in animals by exposure of newborn to a foreign antigen. Depending on the dose and timing of the antigenic challenge, tolerance has been reported to result in clonal deletion, anergy or active suppression. In this latter case, regulatory T cells prevent autoimmunity by suppressing the reactivity of pathogenic self-reactive T cells. We have previously reported the generation of a neonatal, mercury-specific, and dominant tolerance to autoimmunity induced by mercury salts in rats. Chronic exposure to mercury salts can lead to SLE-like autoimmune responses, mediated by autoreactive CD4+ Th2 cells, that regulate and are followed by a resistant state mediated by protective CD8+ T cells. The aim of the study was to compare the resistance to the neonatal tolerance to mercury disease, and to further characterize the CD8+ T cells endowed with regulatory capacity in the neonatal tolerance model. We report here that resistance to mercury disease is long lasting and not mercury-specific, suggesting that different CD8+ T cells are involved in resistance and neonatal tolerance, and that regulatory CD8+ Tc1 cells generated in tolerance are required to control the CD8- cell population from developing Th2-mediated autoimmunity. Upon mercury recall, CD8+ CD45RC(high) T cells, that represent the Tc1 subset in the rat, expanded and were polarized towards IFNgamma production. Interestingly, identical results were obtained with the CD8+ CD25+T cell population. Substantial amounts of FasL gene expression were detected in CD8+ T lymphocytes upon recall with the tolerogen. AICD may be one of the regulatory mechanisms used by these regulatory CD8+ Tc1 cells that control neonatal tolerance to a Th2-mediated autoimmune disorder.

The humoral immune response to macrocyclic chelating agent DOTA depends on the carrier molecule.

UNLABELLED: The chelating agent 1,4,7,10-tetraazacyclododecane-N,N', N",N"'-tetraacetic acid (DOTA) is used to label monoclonal antibodies (mAbs) and peptides with (90)Y. DOTA allows the generation of clinically useful stable metallic radioconjugates for the treatment of a variety of tumors, but its immunogenicity has remained controversial. In this study, we evaluated the immune response to DOTA in a preclinical mouse model and in patients entered in a clinical trial. METHODS: Sera were obtained from BALB/c mice injected intraperitoneally or subcutaneously with different doses and formulations of syngeneic and xenogeneic mAbs or peptide (murine mAb Mov19 [mM19]; its chimeric version; murine V/human C ChiMov19 [cM19]; or Tyr(3)-octreotide)-DOTA conjugates. Sera from patients with neuroendocrine tumors, enrolled in a protocol for somatostatin receptor-mediated radionuclide therapy with (90)Y-DOTA-D-Phe(1)-Tyr(3)-octreotide (DOTATOC), were also collected before and after each treatment. Levels and specificity of antibody response to relevant (Mov19, ChiMov19, or Tyr(3)-octreotide) and nonrelevant (human serum albumin) DOTA targets were tested by enzyme-linked immunosorbent assay and competition assays. An anti-DOTA mAb (IgG1) derived from a ChiMov19-DOTA immunized mouse was used, in a competitive radioimmunoassay, to determine the efficiency of DOTA presentation on the different carriers. RESULTS: Depending on the immunogenicity and dosage of the mAb, a specific anti-DOTA response was revealed in the preclinical system. However, DOTA-peptide conjugate induced no immune-detectable response against either chelator or carrier. DOTA was poorly presented on small peptides, as determined using the anti-DOTA mAb. CONCLUSION: A humoral response against DOTA is possible, but only as a consequence of the response elicited against the carrier. Octreotide was not immunogenic. Thus, (90)Y-DOTATOC can be considered a safe and useful tool for receptor-mediated radionuclide therapy of somatostatin receptor-overexpressing tumors.

The balance between CD45RChigh and CD45RClow CD4 T cells in rats is intrinsic to bone marrow-derived cells and is genetically controlled.

The level of CD45RC expression differentiates rat CD4 T cells in two subpopulations, CD45RC(high) and CD45RC(low), that have different cytokine profiles and functions. Interestingly, Lewis (LEW) and Brown Norway (BN) rats, two strains that differ in their ability to mount type 1 and type 2 immune responses and in their susceptibility to autoimmune diseases, exhibit distinct CD45RC(high)/CD45RC(low) CD4 T cell ratios. The CD45RC(high) subpopulation predominates in LEW rats, and the CD45RC(low) subpopulation in BN rats. In this study, we found that the antiinflammatory cytokines, IL-4, IL-10, and IL-13, are exclusively produced by the CD45RC(low) CD4 T cells. Using bone marrow chimeras, we showed that the difference in the CD45RC(high)/CD45RC(low) CD4 T cell ratio between naive LEW and BN rats is intrinsic to hemopoietic cells. Furthermore, a genome-wide search for loci controlling the balance between T cell subpopulations was conducted in a (LEW x BN) F(2) intercross. Genome scanning identified one quantitative trait locus on chromosome 9 (approximately 17 centiMorgan (cM); log of the odds ratio (LOD) score 3.9). In addition, two regions on chromosomes 10 (approximately 28 cM; LOD score 3.1) and 20 (approximately 40 cM; LOD ratio score 3) that contain, respectively, a cytokine gene cluster and the MHC region were suggestive for linkage. Interestingly, overlapping regions on these chromosomes have been implicated in the susceptibility to various immune-mediated disorders. The identification and functional characterization of genes in these regions controlling the CD45RC(high)/CD45RC(low) Th cell subpopulations may shed light on key regulatory mechanisms of pathogenic immune responses.

Phage library-derived human anti-TETA and anti-DOTA ScFv for pretargeting RIT.

Pretargeting techniques have promise for radioimmunotherapy (RIT) in cancer because of the potential for markedly increasing the therapeutic ratio. Human antibody fragments can be retrieved from phage libraries and used to realize this potential. The library can be used to select the genetic material required to generate molecules with binding sites for both radiochelates and tumor antigens. In this study, human anti-chelate scFvs (single-chain fragments) for two metal chelates (Cu-TETA and Y-DOTA) were selected from a large, naive human scFv library. These anti-chelate scFvs were intended to serve as one arm of bispecific pretargeting molecules and to bind radiochelates given subsequently as Cu-67-TETA or Y-90-DOTA. Phage that displayed the anti-chelate scFv were selected by absorption to antibody (Lym-1) bound Cu-TETA or Y-DOTA. Enzyme-linked immunosorbent assays (ELISA) were performed to assess the intensity and specificity of phage binding to the specific chelate. Ninety-six clones demonstrating metal chelate binding seven times greater than to Lym-1 alone were chosen for diversity analysis. BstN I restriction digests were performed on DNA from these clones. Twenty-three and 43 different DNA fingerprint patterns were identified for anti-TETA and anti-DOTA clones, respectively. DNA sequencing of 39 anti-TETA clones for 23 different BstN I fingerprint patterns revealed 22 distinct sequences. Eleven of the anti-TETA clones were selected for further study. Five hundred to 1000 microg (100 to 320 microg per liter of culture) of purified scFv was produced from each of the 11 anti-TETA clones. Preliminary studies by BIAcore demonstrated evidence of 25- to 200-nM affinities. Comparable examination of the anti-DOTA clones is in progress. This study provides evidence that human scFv against unique synthetic targets can be readily selected from a large, naive human immunoglobulin phage library. Selections against metal chelated antibodies provided a wealth of scFvs with diverse binding affinities useful for engineering molecules for pretargeting RIT.

New anti-Cu-TETA and anti-Y-DOTA monoclonal antibodies for potential use in the pre-targeted delivery of radiopharmaceuticals to tumor.

Monoclonal antibodies were raised against yttrium(III)-1, 4, 7, 10-tetraazacyclododecane-N,N',N''N'''--tetraacetic acid (Y-DOTA) and copper(II)-1, 4, 8, 11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (Cu-TETA). Four hybridomas with high Y-DOTA binding activity and one hybridoma with Cu-TETA activity were selected. MAbs were purified from mouse ascites by Protein A affinity chromatography and characterized. Affinity constants were determined by equilibrium dialysis and the highest affinity Y-DOTA MAb (K(aff) = 1.9 x 10(8) M(-1)) was further characterized by competitive ELISA. Gd-DOTA competed as well as Y-DOTA, whereas In-DOTA required 740x higher concentrations for 50% inhibition of this Y-DOTA MAb binding to human serum albumin-Y-DOTA-coated microtiter plates. These anti-metal chelate MAbs have potential use as vehicles for the pretargeted delivery of radiometal chelates to tumors.

The effect of concurrent lead and cadmium exposure on the cell-mediated immune response in goats.

Cell-mediated immune response was monitored by cutaneous hypersensitivity reaction (CHR) to 2-4 dinitrochlorobenzene in goats given lead or cadmium alone or in combination. Twenty goats were divided into 4 groups of 5 animals each. Group A served as control whereas Groups B, C and D were given po doses of 50 mg lead acetate/kg body weight, 10 mg cadmium chloride/kg body weight or 50 mg lead acetate/kg body weight + 10 mg cadmium chloride/kg body weight, respectively, for 42 d. Primary sensitization was done on day 27 followed by a challenge dose after 14 d. Elicitation of CHR, as measured by average increase in skin thickness, was suppressed significantly in goats of all dosed groups. Suppression was more in the cadmium-dosed than in the lead or lead + cadmium dosed goats. Histopathology demonstrated reduced intensity of cellular reactions in the cadmium and lead + cadmium-dosed animals.