GSK2245035, a TLR7 agonist, Does Not Increase Pregnancy Loss in Cynomolgus Monkeys.

GSK2245035, a small molecule Toll-like Receptor 7 (TLR7) agonist developed for immunomodulatory treatment for allergic airways disease, aimed to reduce Th2 and enhance Th1/Treg responses to aeroallergens via the local induction of type I interferons (IFNs). GSK2245035 demonstrated selectivity for potent release of type I IFNs compared to TNF-α and IL-6, with dose dependent increases in the interferon inducible chemokine, IP-10, in the nasal compartment. Implantation and parturition require pro-inflammatory processes including IFNs, Interferon Stimulated Genes, TNFα and IP-10 while pregnancy requires immune regulation to maintain maternal fetal immune tolerance, and recombinant type I IFNs induced abortions in monkeys. Due to its mechanism of action, GSK2245035 was studied at pharmacologically and clinically relevant doses in a monkey pregnancy model. Monkeys received 0, 3 or 30 ng/kg/week GSK2245035 intranasally once weekly, from Day 20 postcoitum through Day 63 postpartum. Although systemic IFN-α and IP-10 levels were approximately 14.8 or 40 -fold (respectively) above predose levels at 3 or 30 ng/kg/week, respectively, there were no effects on pregnancy and infant outcome. Non-adverse effects included increased incidence of nasal discharge, increased maternal body temperature at 30 ng/kg/week and dose-dependent increases in maternal IP-10 and IFN-α and decreased infant anti-KLH IgM and IgG titers following KLH immunization at ≥3 ng/kg/week, relative to controls. Potentially, lower IFN-α and IP-10 levels as well as once-weekly intranasal dosing vs daily subcutaneous or intramuscular dosing with recombinant type I IFNs could explain the lack of pregnancy effects; however, there was an undesired impact on offspring immune function.

HESI/FDA workshop on immunomodulators and cancer risk assessment: Building blocks for a weight-of-evidence approach.

Profound immunosuppression (e.g., AIDS, transplant therapy) is epidemiologically associated with an increased cancer risk, and often with oncogenic viruses. It is currently unclear how broadly this association translates to therapeutics that modulate immunity. A workshop co-sponsored by the FDA and HESI examined how perturbing the immune system may contribute to carcinogenesis, and highlighted priorities for improving non-clinical risk assessment of targeted immunomodulatory therapies. Conclusions from the workshop were as follows. 1) While profound altered immunity can promote tumorigenesis, not all components of the immune system are equally important in defense against or promotion of cancer and a similar cancer risk for all immunomodulatory molecules should not be assumed. 2) Rodent carcinogenicity studies have limitations and are generally not reliable predictors of cancer risk associated with immunosuppression. 3) Cancer risk needs to be evaluated based on mechanism-based weight-of-evidence, including data from immune function tests most relevant to tumor immunosurveillance or promotion. 4) Information from nonclinical experiments, clinical epidemiology and immunomodulatory therapeutics show that immunosurveillance involves a complex network of cells and mediators. To support a weight-of-evidence approach, an increased focus on understanding the quantitative relationship between changes in relevant immune function tests and cancer risk is needed.

Translational development of an ADAMTS-5 antibody for osteoarthritis disease modification.

OBJECTIVE/METHOD: Aggrecanase activity, most notably ADAMTS-5, is implicated in pathogenic cartilage degradation. Selective monoclonal antibodies (mAbs) to both ADAMTS-5 and ADAMTS-4 were generated and in vitro, ex vivo and in vivo systems were utilized to assess target engagement, aggrecanase inhibition and modulation of disease-related endpoints with the intent of selecting a candidate for clinical development in osteoarthritis (OA). RESULTS: Structural mapping predicts the most potent mAbs employ a unique mode of inhibition by cross-linking the catalytic and disintegrin domains. In a surgical mouse model of OA, both ADAMTS-5 and ADAMTS-4-specific mAbs penetrate cartilage following systemic administration, demonstrating access to the anticipated site of action. Structural disease modification and associated alleviation of pain-related behavior were observed with ADAMTS-5 mAb treatment. Treatment of human OA cartilage demonstrated a preferential role for ADAMTS-5 inhibition over ADAMTS-4, as measured by ARGS neoepitope release in explant cultures. ADAMTS-5 mAb activity was most evident in a subset of patient-derived tissues and suppression of ARGS neoepitope release was sustained for weeks after a single treatment in human explants and in cynomolgus monkeys, consistent with high affinity target engagement and slow ADAMTS-5 turnover. CONCLUSION: This data supports a hypothesis set forth from knockout mouse studies that ADAMTS-5 is the major aggrecanase involved in cartilage degradation and provides a link between a biological pathway and pharmacology which translates to human tissues, non-human primate models and points to a target OA patient population. Therefore, a humanized ADAMTS-5-selective monoclonal antibody (GSK2394002) was progressed as a potential OA disease modifying therapeutic.

Immunomodulatory effects of anti-CD4 antibody in host resistance against infections and tumors in human CD4 transgenic mice.

Anti-CD4 antibodies, which cause CD4(+) T-cell depletion, have been shown to increase susceptibility to infections in mice. Thus, development of anti-CD4 antibodies for clinical use raises potential concerns about suppression of host defense mechanisms against pathogens and tumors. The anti-human CD4 antibody keliximab, which binds only human and chimpanzee CD4, has been evaluated in host defense models using murine CD4 knockout-human CD4 transgenic (HuCD4/Tg) mice. In these mice, depletion of CD4(+) T cells by keliximab was associated with inhibition of anti-Pneumocystis carinii and anti-Candida albicans antibody responses and rendered HuCD4/Tg mice susceptible to P. carinii, a CD4-dependent pathogen, but did not compromise host defense against C. albicans infection. Treatment of HuCD4/Tg mice with corticosteroids impaired host immune responses and decreased survival for both infections. Resistance to experimental B16 melanoma metastases was not affected by treatment with keliximab, in contrast to an increase in tumor colonization caused by anti-T cell Thy1.2 and anti-asialo GM-1 antibodies. These data suggest an immunomodulatory rather than an overt immunosuppressive activity of keliximab. This was further demonstrated by the differential effect of keliximab on type 1 and type 2 cytokine expression in splenocytes stimulated ex vivo. Keliximab caused an initial up-regulation of interleukin-2 (IL-2) and gamma interferon, followed by transient down-regulation of IL-4 and IL-10. Taken together, the effects of keliximab in HuCD4/Tg mice suggest that in addition to depleting circulating CD4(+) T lymphocytes, keliximab has the capability of modulating the function of the remaining cells without causing general immunosuppression. Therefore, keliximab therapy may be beneficial in controlling certain autoimmune diseases.

Highly related immunoglobulin light chain sequences in different multiple sclerosis patients.

Although immunoglobulin G and free light (L) chains of oligoclonal origin in cerebrospinal fluid (CSF) are the most common immunologic abnormalities in multiple sclerosis (MS), it is unknown whether homologous CSF L chain sequences are present in different individuals with MS. Using Southern blotting, a particular kappa (kappa) L chain variable region (V) probe was recently found to hybridize to Vkappa cDNA from CSF B cells from almost one half of the MS patients tested but only 10% of normal or other neurologic disease controls [Zhou, S.-R., Maier, C.C., Mitchell, G.W., LaGanke, C.C., Blalock, J.E., Whitaker, J.N., 1998. A cross-reactive idiotope in cerebrospinal fluid cells in multiple sclerosis: further evidence for the role of myelin basic protein. Neurology 50, 411-417.] Here, we report that this likely results from remarkable sequence similarity in certain Vkappa from CSF B cells from different individuals with MS. The high degree of sequence homology even extended to all three complementarity determining regions (CDR) which in part form an antibody combining site. In addition, marked sequence homology was observed between the light chains from the MS patients and those from certain mouse antibodies against myelin basic protein (MBP). The results establish, in principle, that the same or very similar kappa light chain variable regions can be shared between CSF B lymphocytes from different individuals with MS as well as with certain antibodies against MBP.

Unique molecular surface features of in vivo tolerized T cells.

Differential expression of surface markers can frequently be used to distinguish functional subsets of T cells, yet a surface phenotype unique to T cells induced into an anergic state has not been described. Here, we report that CD4 T cells rendered anergic in vivo by superantigen can be identified by loss of the 6C10 T cell marker. Inoculation of Vbeta8.1 T cell antigen receptor (TCR) transgenic mice with a Vbeta8.1-reactive minor lymphocyte-stimulating superantigen (Mls-1(a)) induces tolerance to Mls-1(a) by clonal anergy. CD4 lymph node T cells from Mls-1(a) inoculated transgenic mice enriched for the 6C10(-) phenotype neither proliferate nor produce interleukin-2 upon TCR engagement, whereas 6C10(+) CD4 T cells retain responsiveness. Analysis of T cell memory markers demonstrate that 6C10(-) T cells remain 3G11(hi) but express heterogeneous levels of CD45RB, CD62L, CD44, and the CD69 early activation marker, suggesting that T cells at various degrees of activation can be functionally anergic. These studies demonstrate that anergic T cells can be purified based on 6C10 expression permitting examination of issues concerning biochemical and biological features specific to T cell anergy.

A cross-reactive anti-myelin basic protein idiotope in cerebrospinal fluid cells in multiple sclerosis.

We wanted to find evidence of antibody to myelin basic protein (MBP) in patients with MS by detecting their shared usage of immunoglobulin genes. As demonstrated by the idiotopes (i.d.) of murine monoclonal antibody to peptides of MBP, there is limited use of the variable (V) region immunoglobulin genes for the immune response in mice to this encephalitogenic protein. Cross-reactive Ids have been detected across different murine strains and shared by T and B cells. One cross-reactive Id, designated as 845D3 Id, is located on the V region of kappa light chains of two murine monoclonal antibodies, one to MBP peptide 80-89 and the other to MBP peptide acetyl 1-9. To examine the occurrence of 845D3 Id in MS, we used the V region of a light chain (VL) of one of the monoclonal antibodies to probe the VL genes expressed in B cells in CSF of 50 patients (31 MS and 19 non-MS). The VL genes expressed in B cells found in CSF were amplified by polymerase chain reaction using universal human V-region primers. The 845D3 Id probe detected the Id+ V region in the CSF of 14 of 31 MS patients, 1 of 9 patients with other neurologic diseases, and 1 of 10 non-neurologic patients. The gene product was more common in but not restricted to CSF with oligoclonal bands. The presence in CSF of MS patients of a cross-reactive Id to different MBP peptides is indicative of an immune response to this encephalitogenic myelin protein in a segment of MS patients. These findings are also evidence for limited usage of V-region Ig genes in the immune response of humans to MBP and the possible importance of an Id network for MBP in demyelinating disease.

Molecular mimicry of carcinoembryonic antigen by peptides derived from the structure of an anti-idiotype antibody.

Our goal was to use carcinoembryonic antigen (CEA) as a target for immunotherapy in CEA-positive cancer patients who are all immune tolerant to the native antigen. We isolated and characterized an anti-idiotype monoclonal antibody 3H1, which mimics a distinct and specific epitope of the Mr 180,000 CEA and can be used as a surrogate for CEA. In Phase Ib clinical trials in a group of 23 advanced colorectal cancer patients, 3H1 induced both humoral and cellular anti-3H1 responses, as well as anti-CEA immunity. To study the cellular immunity invoked by 3H1 at the molecular level, we have cloned and sequenced the cDNAs encoding the variable heavy and light chains of 3H1 and deduced the amino acid sequences of the encoded proteins. To identify any cross-reactive peptides of 3H1 and CEA, we compared the amino acid sequences of 3H1 with those of CEA and found several regions of homology in 3H1 heavy and light chain variable domains, as well as in the framework regions. To search for potential cross-reactive T-cell epitopes, a number of peptides were synthesized based on 3H1/CEA homology and were used as stimulants in cell proliferation assays, using peripheral blood mononuclear cells from a group of 3H1-immunized CEA-positive cancer patients in the adjuvant setting. Two partially homologous peptides, designated LCD-2 (from 3H1) and CEA-B (from CEA), were identified in 10 of 21 adjuvant patients by strong proliferation responses (stimulation index, 3-50-fold), which were extensively studied in five of these individuals over an extended period of time (12-24 months). We saw no correlation with the MHC class I haplotype of the patients. Analysis of the subtype of the responding T cells demonstrated that primarily CD4+ T cells were stimulated by both 3H1 and 3H1-derived peptides. Interleukin 2, interleukin 4, and IFN-gamma were assayed in the culture medium of peripheral blood mononuclear cells stimulated with 3H1, CEA, and LCD-2 to determine the T-cell helper subset induced by these stimulants. The in vitro responses were mainly associated with secretion of IFN-gamma, which suggested that the induced T cells were most likely CD4+ Th1 type. Future studies will include the design of second-generation LCD-2 and CEA peptides to further enhance antigenicity, to characterize the responding T-cell populations more fully, and to test refined peptides for immunogenicity.

Biochemical features of anergic T cells.

T cell anergy is a functionally defined state of hyporesponsiveness in which T cells neither proliferate nor produce IL2 following subsequent TCR ligation. Recent biochemical data from in vitro studies suggest that anergic cells do not utilize all of the signaling pathways normally initiated by TCR triggering. These findings appear to hold true for T cells rendered anergic in vivo, as well; however, biochemical studies on clonal anergy in vivo have been limited by the inability to recover a homogeneous population of anergic T cells. Here we review progress on TCR mediated signaling pathways as well as the description of surface marker phenotypes specific to T cell anergy.

Synthetic CD4 exocyclic peptides antagonize CD4 holoreceptor binding and T cell activation.

We have developed peptide analogs to analyze precise human CD4 substructures involved in MHC class II binding. Forms of the complementarity determining-like regions (CDRs) of the D1 domain of human CD4 were reproduced as synthetic aromatically modified exocyclic (AME) analogs and tested for their ability to block CD4-MHC II interactions and T cell activation. The exocyclic derived from CDR3 (residues 82-89) of human CD4, which specifically associated with CD4 on the T cell surface to create a heteromeric CD4 complex, blocked IL-2 production and antagonized the normal function of the CD4 receptor. The approach of creating novel synthetic antagonistic receptor complexes may represent a new receptor specific pharmaceutical approach to modulate biological function.

Murine V lambda x and V lambda x-containing antibodies bind human myelin basic protein.

Myelin basic protein (MBP) is highly immunogenic and a known autoantigen capable of inducing experimental allergic encephalomyelitis (EAE), the animal model of multiple sclerosis. We have previously described a murine monoclonal antibody (mAb), F28C4, directed against the encephalitogenic MBP peptide acetyl (Ac) 1-9, which contains a V lambda x light chain. Considering the rarity of V lambda x usage, we determined whether other Abs having V lambda x light chains shared similar antigen (Ag) specificity. We screened a panel of V lambda x-containing monoclonal and polyclonal Abs, of unknown specificity for reactivity with MBP. All such Ab, but not heavy chain isotype matched controls, bound MBP but were not polyreactive with other potential self Ags. The binding of a recombinant form of V lambda x alone to MBP demonstrated the important contribution of the V lambda x light chain to the reaction. With the exception of mAb F28C4 which recognizes MBP Ac1-9, the epitope specificity of all other V lambda x-bearing Abs was localized to MBP residues 25-34. These results demonstrate a unique association between V lambda x expression and MBP reactivity. Given that V lambda x shares sequence homology with T cell receptors (TCR) from encephalitogenic T lymphocytes, these results imply a potential role for V lambda x in the pathogenesis of EAE.

Identification of interactive determinants on idiotypic-anti-idiotypic antibodies through comparison of their hydropathic profiles.

We have written a computer program to aid in the identification of interaction sites between proteins. The program compares the hydropathic profiles of the two interacting proteins and reports sites, demonstrating an exact pattern of inverted hydropathy. If these regions are surface accessible in the folded proteins, they are considered putative binding or docking sites and can be tested as such. In this report, we apply this program to the localization of residues involved in the anti-idiotope of a monoclonal antibody (mAb), F30C7. The anti-idiotope of F30C7 partially resembles the structure of the peptide antigen, human myelin basic protein (MBP) acetyl 1-9, used to elicit the idiotope bearing mAbs (Ab1). The sequences of F30C7 variable regions are compared to the variable regions of Ab1, as well as to the peptide antigen used to elicit F30C7. Sites of hydropathic complementarity in F30C7 with Ab1 that also have sequential homology with MBP 1-9 were located, and a synthetic peptide designed from these sequences was found to structurally resemble MBP 1-9 in that it: (i) inhibited Ab1 binding to MBP 1-9 and (ii) partially inhibited the binding of F30C7 to Ab1. Thus the portion of the anti-idiotope of F30C7 resembling MBP 1-9 was determined with the aid of this program. Other hits between F30C7 and Ab1 also occurred, and future studies will determine whether or not these sites might further contribute to the anti-idiotope.

PCR-based cloning, sequencing, and exon mapping of lymphocyte-derived neuroendocrine peptides.

In this report a procedure for the analysis of mRNA expression in cells of limited availability by the reverse transcriptase-polymerase chain reaction (RT-PCR) method is described. The cells are lysed with Nonidet P-40, and the mRNA in the lysate is used directly as template for the cDNA synthesis reaction. Target cDNA is then amplified by PCR, and the products can be analyzed that same day by agarose gel electrophoresis. The oligonucleotide primers used for amplification are designed to include restriction sites to facilitate cloning for subsequent sequencing. We have demonstrated that luteinizing hormone-releasing hormone mRNA can be amplified from the hypothalamus and thymus of a 7-day rat pup, in which the starting cell number was limited. Furthermore, exon usage by target cDNA in different cell types can be easily determined by amplifying with exon-specific primers. Proopiomelanocortin (POMC) mRNA expressed in the pituitary utilizes all three exons, while a majority of POMC mRNA expressed in lymphocytes lacks exons 1 and 2. Thus, this provides an extremely rapid and sensitive means not only for analyzing mRNA expression but also for differential exon usage.

A V lambda x-bearing monoclonal antibody with similar specificity and sequence to encephalitogenic T cell receptors.

The fine specificity of mAb F28C4 to myelin basic protein (MBP), acetyl residues 1-9, has been compared with the previously described specificity of an encephalitogenic T cell clone, PJR-25. F28C4 has been found to express a cross-reactive idiotope (CRI) that is shared with MBP acetyl peptide 1-9-specific TCR. The CRI seems to be located at or near the Ag-combining site of F28C4 and the TCR and, thus, might possibly result from overlapping epitope specificity. We tested the fine epitope specificity of F28C4 by using alanine-substituted peptide analogues and found that residues critical for TCR recognition, Cln3 and Pro6, are also necessary for F28C4 recognition. By using nuclear magnetic resonance, we found that the MBP acetyl peptide 1-9 binds F28C4 in an extended conformation and that the central residues are more tightly bound than the terminal residues, much like the MBP-TCR interaction. Furthermore, sequence homology (75% overall) was found between the regions that contained CDR3 of F28C4 VL and VH and the VDJ junction of the TCR V beta. This homology is not shared by other Ig CDR3 regions and arises, in part, because F28C4 uses an unusual V lambda light chain, V lambda x. Thus, F28C4 shares a CRI with the TCRs, possibly as a result of having similar fine epitope specificity and sequence homology. The anti-CRI mAb can down-modulate experimental allergic encephalomyelitis; thus, it is possible that Abs that are similar to F28C4 may play an important immunoregulatory role in experimental allergic encephalomyelitis in vivo.

The structure of a myelin basic protein-associated idiotope.

A cross-reactive idiotope (CRI) has been previously described on monoclonal antibodies (mAbs) specific for encephalitogenic peptides from myelin basic protein (MBP). The anti-CRI mAb, F25F7, binds an idiotope (Id) localized to the light chains of an anti-MBP peptide 1-9 mAb, denoted F23C6, and an anti-MBP peptide 80-89 mAb, denoted 845D3. It is the purpose of this study to further delineate the CRI being recognized by F25F7. To this end, we have found a structural correlation between the CRI and the antigen, a small synthetic peptide, denoted PBM 9-1, used to elicit the anti-Id mAb. Sequence comparison between the light chain of F23C6 and PBM 9-1 reveals a region of homology in CDR 2/FWK 3. The configuration of this site in the VL, as determined by comparison with a mAb, HyHEL-10, whose structure has been determined and is 97% homologous to the light chain of F23C6, conforms to the rules used to define antigenic determinants or Ids. A synthetic peptide having the F23C6 VL CDR 2/FWK 3 sequence inhibited the binding of F25F7 to F23C6 and 845D3. Taken together, these data suggest the Id recognized by F25F7 is defined, in part, by the PBM 9-1-like sequence of F23C6.

Thymocytes express a mRNA that is identical to hypothalamic luteinizing hormone-releasing hormone mRNA.

1. A luteinizing hormone-releasing hormone (LHRH)-like molecule produced by thymocytes is similar to hypothalamic LHRH in both bioactivity and antigenicity. 2. We determined whether this thymic LHRH is identical to or only homologous with hypothalamic LHRH by synthesizing and sequencing the cDNA of rat thymus LHRH. 3. The thymocyte and hypothalamic LHRH cDNAs are identical, indicating, that the amino acid sequences of LHRH produced in the hypothalamus and the immune system are also identical. 4. This is the first report showing conclusively that cell of the immune system transcribe the authentic mRNA for a hypothalamic releasing factor, LHRH.