Jennifer L. Rojko

Correlation of Toxicity and Pharmacokinetic Properties of a Phosphorothioate Oligonucleotide Designed to Inhibit ICAM-1

ISIS 2302 is a phosphorothioate oligodeoxynucleotide with a sequence complementary to the mRNA of human intercellular adhesion molecule 1 (ICAM-1). Hybridization of ISIS 2302 to the mRNA inhibits expression of the ICAM-1 protein in response to inflammatory stimuli. A murine active antisense oligonucleotide, ISIS 3082, has been used for in vivo pharmacology studies and has anti-inflammatory activity in models of organ transplant rejection, ulcerative colitis, and collagen-induced arthritis at doses ranging from 0.03 to 5 mg/kg. The safety assessment for ISIS 2302 includes general toxicity studies up to 6 mo in duration in mice and monkeys, genetic toxicity studies, and reproductive/fertility studies. ISIS 3082 was examined in parallel with ISIS 2302 in mouse toxicity and reproductive studies. The toxicities observed following systemic administration of ISIS 2302 and ISIS 3082 were similar and consistent with those observed for other compounds in this chemical class and, therefore, are independent of the suppression of ICAM-1 expression. Toxicokinetic evaluation demonstrated that toxicities occurred in organs containing the highest concentrations of ISIS 2302. Evidence of immune stimulation, including dose-dependent splenomegaly, lymphoid hyperplasia, and multiorgan mixed mononuclear cell infiltrates, was the most common finding in rodent studies. Monkeys were much less sensitive than mice to immune stimulation. Kidney contained the highest concentrations of ISIS 2302. Morphologic changes observed in kidney included atrophic and regenerative changes in proximal tubular epithelium; however, there was no evidence of functional abnormalities. Additional histologic changes noted in proximal tubular epithelium included basophilic granules, which were reflective of oligonucleotide distribution and uptake in these cells. Liver also contained high concentrations of oligonucleotide, which were associated with Kupffer cell hypertrophy in mice. Changes in serum transaminases, cholesterol, and triglycerides were reflective of hepatic alterations. In monkeys, high concentrations of oligonucleotide caused a transient increase in clotting times and activation of the alternative complement pathway. All toxicities associated with ISIS 2302 were reversible and occurred at doses well above those required for pharmacologic activity or currently used in clinical trials. In addition, there has been no evidence of genetic toxicity associated with ISIS 2302, and no changes in reproductive performance, fertility, or fetal development have been noted in animals treated with ISIS 2302 or ISIS 3082.

Partial dissociation of subgroup C phenotype and in vivo behaviour in feline leukaemia viruses with chimeric envelope genes

Feline leukaemia viruses (FeLVs) are classified into subgroups A, B and C by their use of different host cell receptors on feline cells, a phenotype which is determined by the viral envelope. FeLV-A is the ubiquitous, highly infectious form of FeLV, and FeLV-C isolates are rare variants which are invariably isolated along with FeLV-A. The FeLV-C isolates share the capacity to induce acute non-regenerative anaemia and the prototype, FeLV-C/Sarma, has strongly age-restricted infectivity for cats. The FeLV-C/Sarma env sequence is closely related to that of common, weakly pathogenic FeLV-A isolates. We now show by construction of chimeric viruses that the receptor specificity of FeLV-A/Glasgow-1 virus can be converted to that of FeLV-C by exchange of a single env variable domain, Vr1, which differs by a three codon deletion and nine adjacent substitutions. Attempts to dissect this region further by directed mutagenesis resulted in disabled proviruses. Sequence analysis of independent natural FeLV-C isolates showed that they have unique Vr1 sequences which are distinct from the conserved FeLV-A pattern. The chimeric viruses which acquired the host range and subgroup properties of FeLV-C retained certain FeLV-A-like properties in that they were non-cytopathogenic in 3201B feline T cells and readily induced viraemia in weanling animals. They also induced a profound anaemia in neonates which had a more prolonged course than that induced by FeLV-C/Sarma and which was macrocytic rather than non-regenerative in nature. Although receptor specificity and a major determinant of pathogenicity segregate with Vr1, it appears that sequences elsewhere in the genome influence infectivity and pathogenicity independently of the subgroup phenotype.

Formation, Clearance, Deposition, Pathogenicity, and Identification of Biopharmaceutical-related Immune Complexes Review and Case Studies

Vascular inflammation, infusion reactions, glomerulopathies, and other potentially adverse effects may be observed in laboratory animals, including monkeys, on toxicity studies of therapeutic monoclonal antibodies and recombinant human protein drugs. Histopathologic and immunohistochemical (IHC) evaluation suggests these effects may be mediated by deposition of immune complexes (ICs) containing the drug, endogenous immunoglobulin, and/or complement components in the affected tissues. ICs may be observed in glomerulus, blood vessels, synovium, lung, liver, skin, eye, choroid plexus, or other tissues or bound to neutrophils, monocytes/macrophages, or platelets. IC deposition may activate complement, kinin, and/or coagulation/fibrinolytic pathways and result in a systemic proinflammatory response. IC clearance is biphasic in humans and monkeys (first from plasma to liver and/or spleen, second from liver or spleen). IC deposition/clearance is affected by IC composition, immunomodulation, and/or complement activation. Case studies are presented from toxicity study monkeys or rats and indicate IHC-IC deposition patterns similar to those predicted by experimental studies of IC-mediated reactions to heterologous protein administration to monkeys and other species. The IHC-staining patterns are consistent with findings associated with generalized and localized IC-associated pathology in humans. However, manifestations of immunogenicity in preclinical species are generally not considered predictive to humans.

Use of Tissue Cross-reactivity Studies in the Development of Antibody-based Biopharmaceuticals: History, Experience, Methodology, and Future Directions

Tissue cross-reactivity (TCR) studies are screening assays recommended for antibody and antibody-like molecules that contain a complementarity-determining region (CDR), primarily to identify off-target binding and, secondarily, to identify sites of on-target binding that were not previously identified. At the present time, TCR studies involve the ex vivo immunohistochemical (IHC) staining of a panel of frozen tissues from humans and animals, are conducted prior to dosing humans, and results are filed with the initial IND/CTA to support first-in-human clinical trials. In some cases, a robust TCR assay cannot be developed, and in these cases the lack of a TCR assay should not prevent a program from moving forward. The TCR assay by itself has variable correlation with toxicity or efficacy. Therefore, any findings of interest should be further evaluated and interpreted in the context of the overall pharmacology and safety assessment data package. TCR studies are generally not recommended for surrogate molecules or for comparability assessments in the context of manufacturing/cell line changes. Overall, the design, implementation, and interpretation of TCR studies should follow a case-by-case approach.

A feline large granular lymphoma and its derived cell line.

SummaryA lymphoma cell line (MCC) was derived from an abdominal mass from a 13-yr-old castrated male cat. The cells resemble natural killer precursor cells, have membrane-bound granules, and are positive for chloroacetate esterase, α-naphthyl butyrate esterase, and tartrate-resistant acid phosphatase activities. The MCC cells are negative for rearranged feline T-cell receptor genes, negative for feline T-cytotoxic antigen, Ia, and surface μ, τ, and lambda chains and do not form E-rosettes. The MCC cell line is negative for the feline leukemia virus (FeLV); e.g., negative for exogenous FeLV (exU3) sequences, negative for cytoplasmic and surface FeLV major core protein of 27 000 daltons (p27) by indirect, immunofluorescence assay, negative for helper FeLV by clone 81 assay, and negative for release of soluble FeLV p27 by enzyme-linked immunosorbent assay. Electron microscopy reveals budding type C retrovirus particles and MCC cells react with anti-RD-114 (anti-endogenous feline retrovirus) reference serum. After in vitro infection, MCC replicate FeLV readily, but replication is noncytopathic.

Immunogenicity/hypersensitivity of biologics.

This continuing education course was designed to provide an overview of the immunologic mechanisms involved in immunogenicity and hypersensitivity reactions following administration of biologics in nonclinical toxicity studies, the methods used to determine whether such reactions are occurring, and the associated clinical and anatomic pathology findings. Hypersensitivity reactions have classically been divided into type I, II, III, and IV reactions; type I and III reactions are those most often observed following administration of biologics. A variety of methods can be used to detect these reactions. Antemortem methods include hematology; detection of antidrug antibodies, circulating immune complexes and complement fragments, and immunoglobulin E in serum; tests for serum complement activity; and evaluation of complement receptor 1 on erythrocytes. Postmortem methods include routine light microscopy and electron microscopy, which can demonstrate typical findings associated with hypersensitivity reactions, and immunohistochemistry, which can detect the presence of immune complexes in tissues, including the detection of the test article. A final determination of whether findings are related to a hypersensitivity reaction in individual animals or across the entire study should rely on the overall weight of evidence, as findings indicative of these reactions are not necessarily consistent across all affected animals.

Lymphocytosis of Large Granular Lymphocytes in Three Dogs

The clinical course and hematologic changes of three dogs with lymphocytosis of cells morphologically resembling large granular lymphocytes are presented. Hemograms from all dogs showed leukocytosis with marked lymphocytosis. Lymphocytes were characterized by abundant basophilic cytoplasm containing distinct granules which varied in size and number. Electron microscopically the granules were membrane-bound with an electron-dense core. Lymphocytes from one dog were positive for alkaline phosphatase activity, and lymphocytes from another dog were positive for alpha naphthyl butyrate esterase activity. Lymphocytes from one dog were positive for surface receptors for the crystalline fraction portion of gamma immunoglobulins.

Characterization and Mitogenesis of Feline Lymphocyte Populations

Feline lymphocyte populations from blood, spleen, lymph node, thymus and bone marrow were examined for the following markers: rosette formation with guinea pig erythrocytes (GPE-T cells); surface feline thymocyte antigen (T cells); surface immunoglobulin (B cells); cytoplasmic immunoglobulin (preB cells, plasma cells); receptors for the Fc portion of IgG (FcyR-positive T and B cells), and the third component of complement (CR-positive cells - primarily B cells). The blastogenic responses of feline lymphocytes from peripheral blood, spleen, lymph node (LN), thymus and marrow were investigated using the following mitogens: Escherichia coli lipopolysaccharide (LPS), dextran sulfate (DxS), concanavalin A (conA), phytohemagglutinin (PHA) and pokeweed mitogen (PWM). Feline T lymphocytes identified by rosette formation with GPE and surface thymocyte antigen were present in thymus, spleen, LN, blood and, rarely, in marrow. T cell subsets, but not B cell subsets, were differentiated according to mitogen activation. Cells responding to PHA were immature, nonrecirculating cells, which were most strongly activated in thymus [stimulation index (SI) = 12], lymph node (SI = 11) and spleen (SI = 6). PWM-responsive cells were relatively mature, recirculating lymphocytes of widespread distribution and blastogenesis was greater in spleen (SI = 36) and LN (SI = 29), followed by thymus (SI = 23) and blood (SI = 15). Con A was a potent mitogen for cells of spleen (SI = 113), blood (SI = 80) and LN (SI = 77), but not thymus (SI = 7), suggesting that the con-A-inducible cell was a mature, recirculating, postthymic cell. Optimal mitogenic response to the B cell mitogen, LPS, was dependent upon increased cell concentrations (3 x 10(5) versus 1 x 10(5) per microtiter well) and increased incubation time (5 days versus 3 days). Surface IgG- and IgM-bearing lymphocytes and CR-bearing cells from spleen and blood were stimulated preferentially. DxS was moderately mitogenic for CR-bearing lymphocytes in spleen (SI = 4), LN (SI = 3) and blood (SI = 3), but not marrow (SI = 1).

In vitro replication and cytopathogenicity of the feline immunodeficiency virus for feline t4 thymic lymphoma 3201 cells

Cytotoxic feline immunodeficiency virus (FIV) infection was established in feline T4 thymic lymphoma 3201 cells with the Petaluma isolate of the feline immunodeficiency virus (FIV-Petaluma). Mg2(+)-dependent, reverse transcriptase (Mg2+ RT) activity and FIV p24/28-positive cells were evident beginning at 18 days postinoculation (dpi). Cell death was observed beginning at 22 dpi, with a maximum of 40% dead (trypan blue dye exclusion at 26 dpi). This cytocidal change was not observed in cultured Crandell feline kidney fibroblasts similarly infected with FIV-Petaluma. The surviving cells grew out and a chronic FIV-producer cell line was established. The 3201 cell-derived FIV (FIV-3201) was far more virulent for FIV-naive feline 3201 cells, with FIV p24/28-positive cells and Mg2+ RT activity first detectable by 4-8 dpi and subsequent loss of cell viability detectable by 8-12 dpi. Maximum kill (40% dead) was observed at 16 dpi. Comparison between viral infectivity of FIV-Petaluma and FIV-3201 for FIV-naive 3201 cells showed an increase of 1 log10 tissue culture infectious doses (TCID50) by amplification/passage in 3201 cells. Cytologic and electron microscopic examination of 3201 cells in FIV-infected cultures showed frequent budding lentiviral particles. This lytic infection system opens the way to the routine detection, isolation, and quantitation of FIV from FIV-infected cats, to the large-scale propagation of the virus, and to a system for evaluation of the mechanisms of FIV lymphocytotoxicity and the development of therapies to counteract lentiviral cytopathicity.

Determinants of Susceptibility and Resistance to Feline Leukemia Virus Infection. I. Role of Macrophages

Abstract The role of autochthonous peritoneal feline macrophages (Mθ) in the age-related resistance of cats to feline leukemia virus (FeLV) was investigated by a study of the functional properties and FeLV susceptibility of Mθ from kittens and adult cats and the effect of hydrocortisone (HC) and silica on Mθ-FeLV interactions. Although the phagocytic functions of isolated Mθ from kittens and adults were equivalent, the mean FeLV susceptibility of Mθ from kittens was five times that of Mθ from adult cats, thus establishing a direct correlation between the age-related susceptibility of cats and Mθ from cats to FeLV. Mθ of viremic cats were found to be infected with FeLV in vivo; virus titers were slightly higher than those obtained after in vitro infection of Mθ. Mθ from cats that had experienced regressive FeLV infection were not significantly more resistant to FeLV infection in vitro than were Mθ from naive adult specific-pathogen-free cats. HC, which has been shown to enhance the in vivo FeLV susceptibility of cats, also enhanced the permissiveness of Mθ from cats to FeLV in vitro (600-fold for Mθ from adult cats and 200-fold for Mθ) from kittens. Mθ permissiveness to FeLV was highly sensitive to HC and occurred in Mθ infected in vivo or in vitro. In parallel with the effect of HC on the natural resistance of cats to FeLV, administration of silica before virus inoculation also markedly enhanced the FeLV susceptibility of adult cats. Silica was toxic for isolated Mθ but not for lymphocytes in vitro, and silica produced monocytopenia and neutrophilia, delayed skin allograft rejection, and augmented feline oncovirus-associated cell membrane antigen antibody responses in vivo. These experiments indicate that Mθ were linked to the natural resistance of cats to FeLV and that the temporary elimination of Mθ functions (e.g., by silica) and/or the conversion of the Mθ-FeLV relationship from a nonpermissive to a permissive state (e.g., by corticosteroids) resulted in failure of early virus containment, in persistent virus amplification in hemolymphatic tissues, and in subsequent FeLV-related proliferative or antiproliferative disease.