A phase I/II pharmacokinetic and pharmacogenomic study of calcitriol in combination with cisplatin and docetaxel in advanced non-small-cell lung cancer.

BACKGROUND: Preclinical studies demonstrated antiproliferative synergy of 1,25-D3 (calcitriol) with cisplatin. The goals of this phase I/II study were to determine the recommended phase II dose (RP2D) of 1,25-D3 with cisplatin and docetaxel and its efficacy in metastatic non-small-cell lung cancer. METHODS: Patients were ≥18 years, PS 0-1 with normal organ function. In the phase I portion, patients received escalating doses of 1,25-D3 intravenously every 21 days prior to docetaxel 75 mg/m(2) and cisplatin 75 mg/m(2) using standard 3 + 3 design, targeting dose-limiting toxicity (DLT) rate <33 %. Dose levels of 1,25-D3 were 30, 45, 60, and 80 mcg/m(2). A two-stage design was employed for phase II portion. We correlated CYP24A1 tagSNPs with clinical outcome and 1,25-D3 pharmacokinetics (PK). RESULTS: 34 patients were enrolled. At 80 mcg/m(2), 2/4 patients had DLTs of grade 4 neutropenia. Hypercalcemia was not observed. The RP2D of 1,25-D3 was 60 mcg/m(2). Among 20 evaluable phase II patients, there were 2 confirmed, 4 unconfirmed partial responses (PR), and 9 stable disease (SD). Median time to progression was 5.8 months (95 % CI 3.4, 6.5), and median overall survival 8.7 months (95 % CI 7.6, 39.4). CYP24A1 SNP rs3787554 (C > T) correlated with disease progression (P = 0.03) and CYP24A1 SNP rs2762939 (C > G) trended toward PR/SD (P = 0.08). There was no association between 1,25-D3 PK and CYP24A1 SNPs. CONCLUSIONS: The RP2D of 1,25-D3 with docetaxel and cisplatin was 60 mcg/m(2) every 21 days. Pre-specified endpoint of 50 % confirmed RR was not met in the phase II study. Functional SNPs in CYP24A1 may inform future studies individualizing 1,25-D3.

A V3 loop haptenic peptide sequence, when tandemly repeated, enhances immunogenicity by facilitating helper T-cell responses to a covalently linked carrier protein.

Subunit immunogens containing tandemly repeated copies of T- and B-cell epitopes have been shown to be more immunogenic than the respective immunogen containing only a single copy of the sequence. It has been unclear, however, whether the increased immunogenicity of a tandemly repeated B-cell epitope necessarily results from increased helper T-cell responses to intrinsic T-cell epitopes in the tandemly repeated sequences, or to neodeterminant T-cell epitopes created at the junction of adjacent repeat sequences. We examined this question by comparing the immunogenicity in mice of two immunogens containing one or eight tandemly repeated copies of an HIV-1 V3 loop haptenic sequence. Our results show that the tandemly repeated haptenic sequence potentiates the immunogenicity of the protein construct, likely through the facilitation of enhanced B-cell interaction with the tandem repeat construct and the consequent elicitation of increased carrier protein-specific helper T-cell responses.

Recovery and biological activity of filgrastim after injection through silicone rubber catheters.

The recovery and biological activity of filgrastim after injection through a silicone rubber catheter were studied. Various volumes of filgrastim injection 300 micrograms/mL (0.17, 0.34, 0.51, 0.68, 0.85, and 1.0 mL) were injected through silicone rubber catheters and collected in glass vials to simulate intravenous bolus injections. The catheters were flushed with 3 mL of 5% dextrose injection before and after the injections. For some catheters, the procedure was repeated to simulate the administration of a second filgrastim dose through the same catheter. The theoretical filgrastim concentration in the expelled fluid was 16.1, 30.5, 43.6, 55.4, 66.2, or 75.0 micrograms/mL (corresponding to the filgrastim injection volumes). Filgrastim concentrations in expelled fluid were measured with an enzyme-linked immunosorbent assay, and biological activity was measured with a mitogenic cell-culture assay. There was > or = 10% loss of filgrastim after the first injection when the injection volume was 0.17, 0.34, or 0.68 mL. Mean drug recovery after the second filgrastim injection exceeded that after the first for all six volumes and was > or = 90% for five volumes. The recovered filgrastim retained all of its activity. A > or = 10% loss of filgrastim occurred for three of six volumes after one injection through a silicon rubber catheter. Recovery was higher after a second injection through the same catheter. Biological activity was not affected.

Toward a vaccine for AIDS: the emergence of immunobiology-based vaccine development.

Over a decade has passed since the identification of the human immunodeficiency virus (HIV) as the causative agent of AIDS. During this time, HIV has been extensively characterized, and a variety of vaccine constructs and strategies have been explored. For the most part, these have been driven by successes of the past with other pathogens, or by novel approaches enabled by technologies of the present. With the maturing of our insights into the immunopathology of HIV and basic immunological mechanisms, we are presented with unprecedented opportunities to rationally develop vaccine approaches strategically designed to counter the immunopathology of HIV. As opposed to absolute prevention of infection, the primary goal of these strategies may be to limit infection and to assure a response to infection that prevents disease and transmission. Thus, such vaccines may find utility in both preventive and therapeutic roles. In this paper, we present the background and current state of immunobiology-driven vaccine development for AIDS.

A vector for facile PCR product cloning and modification generating any desired 4-base 5' overhang: pRPM.

A pair of plasmids were developed for cloning PCR products in order to facilitate the preparation of products with 5' overhangs consisting of any desired 4-nucleotide sequence. These vectors allow DNA to be cloned into a unique restriction site by blunt-end ligation or by AT-cloning. The cloned DNA is subsequently excised using class IIS restriction enzyme sites flanking the insert yielding a fragment that is entirely free of vector sequences. These enzymes recognize sequences in the vector but "reach-over" the junction to cut within the insert thereby generating a 4-base 5' overhang sequence determined by the 5' sequences in the insert. Thus, in cases where the insert was originally generated by PCR, the overhangs are specified by the primer. More generally, these vectors offer a unique capability for the reversible cloning of any blunt DNA fragment because excision and fill-in reactions precisely regenerate the original blunt fragment regardless of its sequence. These "reach-over" product modification vectors represent general and flexible tools for the generation of fragments for use in engineering DNA constructs.

Enhancement of lymphocyte proliferation assays by use of serum-free medium.

Fetal bovine serum (FBS)-supplemented cell culture medium has been the standard medium used in assays of murine lymphocyte proliferation. While FBS allows cell growth and viability, it requires screening and contains uncharacterized elements which may yield inconsistent results from batch to batch. It also may include growth factors and immunologically reactive proteins that obscure assays for specific responses because of high background proliferation. Recently, chemically defined serum-free media have become available for human lymphocyte culture. We compared several of these media against FBS-supplemented medium and found that one of them, AIM V (Gibco), produced a marked increase in the ratio of positive to background counts in [3H]thymidine incorporation assays of antigen-specific proliferation. Similar results were obtained when responses to mitogenic lectins were examined. AIM V supported proliferation of lymphocytes in a variety of haplotypes, it supported MHC-specific proliferation in response to soluble antigens in a manner consistent with previous reports, and it supported proliferation in response to alloantigen as displayed in the mixed lymphocyte culture. A high ratio of positive to background counts was consistently observed. The results indicate that this serum-free medium enhances the analytic power of proliferation assays.

Hsp27 is a mediator of sustained smooth muscle contraction in response to bombesin.

We have identified the low MW 27 kD heat shock protein as a major phosphoprotein constituent of smooth muscle and have investigated its potential role in agonist induced smooth muscle contraction. The neuropeptides bombesin and substance P, which are present in neurons of the anorectal region, induce contraction of isolated smooth muscle cells from this region by activating different intracellular pathways. Substance P-induced contraction is 1,4,5-inositol trisphosphate (IP3)/calmodulin dependent, while contraction induced by bombesin is mediated by a protein kinase C (PKC)-dependent pathway. The sustained contraction induced by bombesin or exogenous PKC was blocked by preincubation of cells with monoclonal antibodies to hsp27, while the transient contraction induced by substance P or IP3 was unaffected by the antibodies. Preincubation with isotype matched control antibodies had no inhibitory effect on contraction induced in response to the agents used. These data support a novel role for hsp27 in the non calmodulin mediated sustained contraction induced by bombesin or PKC.

Priming with T helper cell epitope peptides enhances the antibody response to the envelope glycoprotein of HIV-1 in primates.

The induction of a memory immune response to HIV, mediated by any kind of effector mechanism, requires the induction of T cell help. In previous studies performed in different murine MHC haplotypes, three immunodominant T cell epitopes (T1, T2, and TH4.1) had been identified in the HIV envelope glycoprotein. Moreover, these peptides were proliferative T cell epitopes in humans. In this study, rhesus monkeys, Macaca mulatta, were primed with these three peptides either in combination or given separately. Half of the monkeys had a proliferative response to one or more of the priming peptide(s). Those monkeys who had a T cell proliferative response also had a high antibody response after one boost with a suboptimal dose of the native protein gp 160, whereas three of four control monkeys who had received only the native protein immunization gave no detectable antibody response, and one displayed a very weak response. For reasons that are unclear, antibodies only to the gp41 portion of gp 160 could be detected in the sera. Thus, the peptides can prime Th cells in primates for an enhanced antibody response on first exposure to the whole protein. The three peptides belong to highly conserved and nonglycosylated regions of the envelope protein. The fact that the peptides acted as immunogenic T cell proliferative and helper epitopes in nonhuman primates is very encouraging for including them in future vaccine studies in humans.

Peptide component vaccine engineering: targeting the AIDS virus.

Most of the successful vaccines developed to date induce protective immunity resembling that produced by natural infection. HIV infection does not induce protective immunity. Thus, previously successful approaches based on live- or killed-virus preparations may not yield an effective and safe AIDS vaccine and many feel that a more highly engineered vaccine will be required. Synthetic peptides represent extremely powerful tools for vaccine research and construct optimization. The theory and practice of vaccine engineering using synthetic peptide components is reviewed with special emphasis on progress towards development of a vaccine for AIDS.

Fine specificity of T cell recognition of the same peptide in association with different I-A molecules.

The fine specificity of the response of T cell clones derived from B10.BR and B10.S congenic mouse strains restricted by I-Ak and I-As molecules, respectively, and which recognize the same 17 amino acid sequence (102-118) of myoglobin, has been investigated and compared with that of T cell clones specific for the same peptide with I-A.d The critical amino acid residues within the 102-118 region of myoglobin required for stimulation of I-Ak-and I-As-restricted T cell clones specific for this determinant were compared using a panel of synthetic peptide analogs. Residues 109, 113, and 116 were critical for stimulation of clones from both haplotypes, although the precise fine specificity varied, even among clones using the same restriction molecule. Residues 109 and 116 are also critical for stimulation of myoglobin-specific I-Ad-restricted clones (Berkower, I., L. A. Matis, G.K. Buckenmeyer, F.R. N. Gurd, D. L. Longo, and J. A. Berzofsky. J. Immunol. 132:1370, 1984). There was also considerable overlap in the size of the minimal determinant necessary for full activity: 106-118 for B10.BR and B10.D2 (Cease, K. B., I. Berkower, J. York-Jolley, and J. A. Berzofsky. J. Exp. Med. 164:1779, 1986) clones and 102-117 for B10.S clones. Despite this similarity in fine specificity, T cell clones were genetically restricted and could not be stimulated with the 102-118 peptide presented by Ia molecules of other haplotypes that could also present this epitope to syngeneic clones. These results suggest that binding of an immunogenic peptide to class II molecules is not sufficient to ensure recognition by a given T cell antigen receptor specific for the peptide, but do not indicate whether the major histocompatibility complex molecules interact directly with the T cell antigen receptor or induce a different recognizable conformation of the peptide.

Identification of proteases that process distinct epitopes on the same protein.

Proteolytic degradation of protein antigens is thought to be a major step in the processing of Ag for presentation to T cells, but the range of proteases involved is unknown. Here we used a large panel of protease inhibitors to determine the role of each of the four classes of proteases in antigen processing. Moreover, we asked whether different proteases were necessary for presentation of different known epitopes, defined by three Th cell clones. For all three epitopes of myoglobin, intracellular thiol proteases such as cathepsins B or L were the only proteases necessary. Furthermore, myoglobin pre-digested with cathepsin B could be presented to all three clones without further processing. Thus, a single protease may be both necessary and sufficient for Ag processing to present the majority of epitopes, at least for myoglobin. This finding provides an explanation of earlier data on the fragments produced from processed myoglobin, and so may contribute to a much needed solution to the long standing problem of predicting where a protein will be cleaved during processing.

T cell multideterminant regions in the human immunodeficiency virus envelope: toward overcoming the problem of major histocompatibility complex restriction.

Helper T cell determinants should be an important component of an anti-human immunodeficiency virus (HIV) vaccine aimed at either antibody or cytotoxic T cell immunity. However, model protein studies have raised concern about the usefulness of any single determinant, because a given determinant is likely to be seen by only a small subset of major histocompatibility complex (MHC) types within the population. Here, we use 44 peptides, including ones predicted and not predicted on the basis of amphipathicity to be potential T cell sites, to locate T cell antigenic determinants recognized by mice of four MHC haplotypes immunized with the whole gp 160 envelope protein. Although the preselection of peptides necessitates caution in a statistical analysis, alpha-amphipathic peptides predominated among sites eliciting the strongest response. Although we have not tested the entire sequence, we have identified six multideterminant regions, in which overlapping peptides are recognized by mice of either three or all four MHC types. Four of the six regions have sequences relatively conserved among HIV-1 isolates. The existence of such multideterminant regions recognized by multiple MHC haplotypes suggests the possibility that use of peptides longer than a minimal determinant and containing several overlapping determinants may be a possible approach to circumvent the serious problem of MHC restriction in peptide vaccines aimed at eliciting T cell immunity.

Therapy of patients with human T-cell lymphotrophic virus I-induced adult T-cell leukemia with anti-Tac, a monoclonal antibody to the receptor for interleukin-2.

Human T-cell lymphotropic virus I (HTLV-I)-induced adult T-cell leukemia (ATL) cells constitutively express interleukin-2 (IL-2) receptors identified by the anti-Tac monoclonal antibody (MoAb), whereas normal resting cells do not. This observation provided the scientific basis for a trial of intravenous anti-Tac in the treatment of nine patients with ATL. The patients did not suffer untoward reactions and did not have a reduction in the normal formed elements of the blood, and only one of the nine produced antibodies to the anti-Tac MoAb. Three patients had transient mixed, partial, or complete remissions lasting from 1 to more than 8 months after anti-Tac therapy, as assessed by routine hematologic tests, immunofluorescence analysis of circulating cells, and molecular genetic analysis of HTLV-I provirus integration and of the T-cell receptor gene rearrangement. The precise mechanism of the antitumor effects is unclear; however, the use of a MoAb that prevents the interaction of IL-2 with its receptor on ATL cells provides a rational approach for the treatment of this malignancy.

Antigenic peptides recognized by T lymphocytes from AIDS viral envelope-immune humans.

T-lymphocyte immunity is likely to be an important component of the immune defence against the AIDS virus, because helper T cells are necessary for the antibody response as well as the cytotoxic response. We have previously predicted two antigenic sites of the viral envelope protein gp120 likely to be recognized by T lymphocytes, based on their ability to fold as amphipathic helices, and have demonstrated that these are recognized by T cells of mice immunized with gp120 (ref. 1). A peptide corresponding to one of these sites can also be induce immunity in mice to the whole gp120 protein. Because many clinically healthy seropositive blood donors have already lost their T-cell proliferative response to specific antigen, we tested the response to these synthetic peptides of lymphocytes from 14 healthy human volunteers who had been immunized with a recombinant vaccinia virus containing the AIDS viral envelope gene and boosted with a recombinant fragment. Eight of the 14 responded to one peptide, and four to the other peptide, not included in the boost. These antigenic sites recognized by human T cells may be useful components of a vaccine against AIDS. We also found a correlation between boosting with antigen-antibody complexes (compared to free antigen) and higher stimulation indices, suggesting a more effective method of immunization.

Influences of antigen processing on the expression of the T cell repertoire. Evidence for MHC-specific hindering structures on the products of processing.

Two lines of evidence in the current study indicate that antigen processing is a major factor, in addition to MHC binding and T cell repertoire, that determines Ir gene responsiveness and epitope immunodominance. First, immunization with synthetic peptides of myoglobin sequences revealed new reactivities that had not appeared after priming with native myoglobin. For example, B10.S mice (H-2S) immune to equine myoglobin predominantly responded to peptide 102-118, whereas there was little, if any, response to this peptide in B10.BR (H-2k) mice immunized with native equine myoglobin. However, after immunization with the 102-118 peptide, both strains responded to the peptide. After in vitro restimulation, B10.BR T cells responded as well as B10.S T cells. Similarly, some individual 102-118-specific T cell clones from mice of both haplotypes showed similar dose responses and fine specificity patterns. Thus, low responsiveness to this site is due neither to a hole in the repertoire nor to a failure to bind to the appropriate MHC molecule. An alternative explanation was suggested by the observation that, whereas B10.S T cells from peptide 102-118-immune mice responded almost as well to whole myoglobin as to the peptide, the B10.BR T cells from peptide immune mice, while responding well to peptide, were poorly stimulated by whole myoglobin. Thus, the product of natural processing of equine myoglobin probably has hindering structures in the regions flanking the core epitope 102-118 that interfere with presentation by I-Ak but not I-AS. The second line of evidence that processing of native myoglobin may influence the apparent specificity of the T cell response was obtained using the I-Ad-restricted sperm whale myoglobin 102-118-specific clone 9.27. This clone discriminated readily between whole sperm whale myoglobin and equine myoglobin, but it did not distinguish between peptides corresponding to 102-118 of the sperm whale and equine sequences. This distinction between equine peptide and native equine myoglobin could be overcome by artificial "processing" of equine myoglobin with cyanogen bromide. In both sets of experiments, F1 APCs that present the same epitope well to T cells of another haplotype failed to overcome the defect, which was therefore not due to the availability of different processed cleavage fragments in APC of different haplotypes, as would be expected if there were MHC-linked processing. Thus, the differential responses to peptides versus native molecule for both I-Ad- and I-Ak-restricted clones appeared to depend on the restricting molecule used.(ABSTRACT TRUNCATED AT 400 WORDS)

An immunodominant epitope of the human immunodeficiency virus envelope glycoprotein gp160 recognized by class I major histocompatibility complex molecule-restricted murine cytotoxic T lymphocytes.

Because cytotoxic T lymphocytes (CTL) may be important for preventing direct cell-to-cell transmission of human immunodeficiency virus (HIV), the agent responsible for acquired immunodeficiency syndrome, we have begun to investigate the epitope specificity and immune response (Ir) gene control of anti-HIV CTL responses in experimental animals. Mice were infected with a recombinant vaccinia virus expressing the HIV gp160 envelope gene, and the primed lymphocytes were restimulated in vitro with a transfected histocompatible cell line expressing the same gene. Our results show that H-2d mice are CTL high responders and H-2k mice are low responders to the HIV gp160 envelope protein under these conditions. Moreover, the H-2d mice respond predominantly to a single immunodominant site represented by a 15-residue synthetic peptide conforming to the amphipathic alpha-helix model of T-cell epitopes and seen by CD4- CD8+ CTL in association with the Dd class I major histocompatibility complex (MHC) molecules. The facts that CTL responses were detected in the context of only one of four class I MHC molecules tested and that the response was limited predominantly to a single epitope indicate that the CTL repertoire elicited by the HIV envelope protein in association with murine class I MHC molecules may be very limited. In addition, this epitope occurs in a highly variable segment of the envelope protein. This puts constraints on the use of a single peptide sequence from this region in a vaccine, as such a vaccine would have to be polyvalent. Nevertheless, this same variability suggests that this region may be under selective pressure from human CTL, and therefore that this site may be immunodominant in humans as well as mice and so of clinical importance in vaccine development.