Fully human CD19-specific chimeric antigen receptors for T-cell therapy.

Impressive results have been achieved by adoptively transferring T-cells expressing CD19-specific CARs with binding domains from murine mAbs to treat B-cell malignancies. T-cell mediated immune responses specific for peptides from the murine scFv antigen-binding domain of the CAR can develop in patients and result in premature elimination of CAR T-cells increasing the risk of tumor relapse. As fully human scFv might reduce immunogenicity, we generated CD19-specific human scFvs with similar binding characteristics as the murine FMC63-derived scFv using human Ab/DNA libraries. CARs were constructed in various formats from several scFvs and used to transduce primary human T-cells. The resulting CD19-CAR T-cells were specifically activated by CD19-positive tumor cell lines and primary chronic lymphocytic leukemia cells, and eliminated human lymphoma xenografts in immunodeficient mice. Certain fully human CAR constructs were superior to the FMC63-CAR, which is widely used in clinical trials. Imaging of cell surface distribution of the human CARs revealed no evidence of clustering without target cell engagement, and tonic signaling was not observed. To further reduce potential immunogenicity of the CARs, we also modified the fusion sites between different CAR components. The described fully human CARs for a validated clinical target may reduce immune rejection compared with murine-based CARs.

Experimental Hendra virus infection of dogs: virus replication, shedding and potential for transmission.

OBJECTIVE: Characterisation of experimental Hendra virus (HeV) infection in dogs and assessment of associated transmission risk. METHODS: Beagle dogs were exposed oronasally to Hendra virus/Australia/Horse/2008/Redlands or to blood collected from HeV-infected ferrets. Ferrets were exposed to oral fluids collected from dogs after canine exposure to HeV. Observations made and samples tested post-exposure were used to assess the clinical course and replication sites of HeV in dogs, the infectivity for ferrets of canine oral fluids and features of HeV infection in dogs following contact with infective blood. RESULTS: Dogs were reliably infected with HeV and were generally asymptomatic. HeV was re-isolated from the oral cavity and virus clearance was associated with development of virus neutralising antibody. Major sites of HeV replication in dogs were the tonsils, lower respiratory tract and associated lymph nodes. Virus replication was documented in canine kidney and spleen, confirming a viraemic phase for canine HeV infection and suggesting that urine may be a source of infectious virus. Infection was transmitted to ferrets via canine oral secretions, with copy numbers for the HeV N gene in canine oral swabs comparable to those reported for nasal swabs of experimentally infected horses. CONCLUSION: HeV is not highly pathogenic for dogs, but their oral secretions pose a potential transmission risk to people. The time-window for transmission risk is circumscribed and corresponds to the period of acute infection before establishment of an adaptive immune response. The likelihood of central nervous system involvement in canine HeV infection is unclear, as is any long-term consequence.

CD19-Targeted chimeric antigen receptor-modified T-cell immunotherapy for B-cell malignancies.

Chimeric antigen receptors (CARs) comprise a tumor-targeting moiety, often in the form of a single chain variable fragment derived from a monoclonal antibody, fused to one or more intracellular T-cell signaling sequences. Lymphodepletion chemotherapy followed by infusion of T cells that are genetically modified to express a CD19-specific CAR is a promising therapy for patients with refractory CD19(+) B-cell malignancies, producing rates of complete remission that are remarkably high in acute lymphoblastic leukemia and encouraging in non-Hodgkin lymphoma and chronic lymphocytic leukemia. Responses are often durable, although additional studies are needed to define the role of CAR-T cell immunotherapy in the context of other treatments. CAR-modified T-cell immunotherapy can be complicated by cytokine release syndrome and neurologic toxicity, which in most cases are manageable and reversible. Here we review recent clinical trial data and discuss issues for the field.

Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo.

Adoptive T-cell therapy with gene-modified T cells expressing a tumor-reactive T-cell receptor or chimeric antigen receptor (CAR) is a rapidly growing field of translational medicine and has shown success in the treatment of B-cell malignancies and solid tumors. In all reported trials, patients have received T-cell products comprising random compositions of CD4(+) and CD8(+) naive and memory T cells, meaning that each patient received a different therapeutic agent. This variation may have influenced the efficacy of T-cell therapy, and complicates comparison of outcomes between different patients and across trials. We analyzed CD19 CAR-expressing effector T cells derived from different subsets (CD4(+)/CD8(+) naive, central memory, effector memory). T cells derived from each of the subsets were efficiently transduced and expanded, but showed clear differences in effector function and proliferation in vitro and in vivo. Combining the most potent CD4(+) and CD8(+) CAR-expressing subsets, resulted in synergistic antitumor effects in vivo. We show that CAR-T-cell products generated from defined T-cell subsets can provide uniform potency compared with products derived from unselected T cells that vary in phenotypic composition. These findings have important implications for the formulation of T-cell products for adoptive therapies.

Development and validation of a 3ABC antibody ELISA in Australia for foot and mouth disease.

OBJECTIVE: To measure the diagnostic performance of an Australian-developed ELISA for the detection of antibodies against the non-structural proteins (NSP) 3ABC of the foot and mouth disease (FMD) virus. DESIGN: Test development and validation study. METHODS: The diagnostic specificity was determined using 2535 sera from naïve animals and 1112 sera from vaccinated animals. Diagnostic sensitivity was calculated from the data for 995 sera from experimentally and field-infected animals from FMD-endemic countries in South East Asia. A commercial ELISA detecting antibodies against FMD virus NSP was used as the reference test to establish relative sensitivity and specificity. Bayesian latent class analysis was performed to corroborate results. The diagnostic window and rate of detection were determined at different times using sera from cattle, sheep and pigs before and after infection, and after vaccination and subsequent infection. Repeatability and reproducibility data were established. RESULTS: At 35% test cut-off, the 3ABC ELISA had an overall diagnostic sensitivity of 91.5% and diagnostic specificity of 96.4%. The diagnostic sensitivity in vaccinated and subsequently infected cattle was 68.4% and diagnostic specificity in vaccinated cattle was 98.0%. CONCLUSIONS: The 3ABC ELISA identified field and experimentally infected animals, as well as vaccinated and subsequently infected animals. Diagnostic sensitivity and specificity estimates for other FMD NSP tests are comparable with the results obtained in this study. This NSP ELISA was found to be 'fit for purpose' as a screening assay at the herd level to detect viral infection and also to substantiate absence of infection.

Henipavirus microsphere immuno-assays for detection of antibodies against Hendra virus.

Hendra and Nipah viruses (HeV and NiV) are closely related zoonotic pathogens of the Paramyxoviridae family. Both viruses belong to the Henipavirus genus and cause fatal disease in animals and humans, though only HeV is endemic in Australia. In general and due to the acute nature of the disease, agent detection by PCR and virus isolation are the primary tools for diagnostic investigations. Assays for the detection of antibodies against HeV are fit more readily for the purpose of surveillance testing in disease epidemiology and to meet certification requirements in the international movement of horses. The first generation indirect ELISA has been affected by non-specific reactions which must be resolved using virus neutralisation serology conducted at laboratory bio-safety level 4 containment (PC4). Recent developments have enabled improvements in the available serology assays. The production of an expressed recombinant truncated HeV G protein has been utilised in ELISA and in Luminex-based multiplexed microsphere assays. In the latter format, two Luminex assays have been developed for use in henipavirus serology: a binding assay (designed for antibody detection and differentiation) and a blocking assay (designed as a surrogate for virus neutralisation). Equine and canine field sera were used to evaluate the two Luminex assays relative to ELISA and virus neutralisation serology. Results showed that Luminex assays can be effective as rapid, sensitive and specific tests for the detection of HeV antibody in horse and dog sera. The tests do not require PC4 containment and are appropriate for high throughput applications as might be required for disease investigations and other epidemiological surveillance. Also, the results show that the Luminex assays detect effectively HeV vaccine-induced antibodies.

Severe Isospora (Cystoisospora) belli Diarrhea Preceding the Diagnosis of Human T-Cell-Leukemia-Virus-1-Associated T-Cell Lymphoma.

Isospora (Cystoisospora) belli diarrhea can sometimes be fulminant in immunocompromised patients. It is endemic in tropical and subtropical areas, and sporadic episodes have been reported in nonendemic areas in nursing homes, day-care centers, and psychiatric institutions. We describe isosporiasis in an HIV-negative Sudanese-American female who presented with a debilitating diarrheal illness and profound weight loss. Isospora belli was detected in her stool by modified acid-fast staining. Serologic testing was negative for HIV but positive for HTLV-1 infection. Treatment with TMP-SMZ led to improvement in her diarrhea which recurred after stopping antibiotics. Subsequently, she developed generalized lymphadenopathy which was diagnosed as ATLL on immunohistochemical staining. Chemotherapy was initiated, but her condition continued to worsen due to persistent diarrhea and resulting profound electrolyte abnormalities. The patient opted for comfort measures and died a few weeks later at a nursing facility. This case emphasizes that the detection of I. belli should trigger testing for HIV, HTLV-1, and other causes of immunocompromise. We suggest that treatment with TMP-SMZ should be initiated and continued for a prolonged period of time in immunocompromised patients with I. belli diarrhea.

HapMap SNP Scanner: an online program to mine SNPs responsible for cell phenotype.

Minor histocompatibility (H) antigens are targets of graft-vs-host disease and graft-vs-tumor responses after human leukocyte antigen matched allogeneic hematopoietic stem cell transplantation. Recently, we reported a strategy for genetic mapping of linkage disequilibrium blocks that encoded novel minor H antigens using the large dataset from the International HapMap Project combined with conventional immunologic assays to assess recognition of HapMap B-lymphoid cell line by minor H antigen-specific T cells. In this study, we have constructed and provide an online interactive program and demonstrate its utility for searching for single-nucleotide polymorphisms (SNPs) responsible for minor H antigen generation. The website is available as 'HapMap SNP Scanner', and can incorporate T-cell recognition and other data with genotyping datasets from CEU, JPT, CHB, and YRI to provide a list of candidate SNPs that correlate with observed phenotypes. This method should substantially facilitate discovery of novel SNPs responsible for minor H antigens and be applicable for assaying of other specific cell phenotypes (e.g. drug sensitivity) to identify individuals who may benefit from SNP-based customized therapies.

A non-human primate model for analysis of safety, persistence, and function of adoptively transferred T cells.

BACKGROUND: Adoptive immunotherapy with antigen-specific effector T-cell (T(E) ) clones is often limited by poor survival of the transferred cells. We describe here a Macaca nemestrina model for studying transfer of T-cell immunity. METHODS: We derived, expanded, and genetically marked CMV-specific CD8(+) T(E) clones with surface markers expressed on B cells. T(E) cells were adoptively transferred, and toxicity, persistence, retention of introduced cell-surface markers, and phenotype of the persisting T cells were evaluated. RESULTS: CD8(+) T(E) clones were efficiently isolated from distinct memory precursors and gene-marking with CD19 or CD20 permitted in vivo tracking by quantitative PCR. CD19 was a more stable surface marker for tracking cells in vivo and was used to re-isolate cells for functional analysis. Clonally derived CD8(+) T(E) cells differentiated in vivo to phenotypically and functionally heterogeneous memory T-cell subsets. CONCLUSIONS: These studies demonstrate the utility of Macaca nemestrina for establishing principles for T-cell therapeutics applicable to humans.

Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells.

Adoptive T cell therapy, involving the ex vivo selection and expansion of antigen-specific T cell clones, provides a means of augmenting antigen-specific immunity without the in vivo constraints that can accompany vaccine-based strategies. A phase I study was performed to evaluate the safety, in vivo persistence, and efficacy of adoptively transferred CD8+ T cell clones targeting the tumor-associated antigens, MART1MelanA and gp100 for the treatment of patients with metastatic melanoma. Four infusions of autologous T cell clones were administered, the first without IL-2 and subsequent infusions with low-dose IL-2 (at 0.25, 0.50, and 1.0 x 10(6) unitsm(2) twice daily for the second, third, and fourth infusions, respectively). Forty-three infusions of MART1MelanA-specific or gp100-specific CD8+ T cell clones were administered to 10 patients. No serious toxicity was observed. We demonstrate that the adoptively transferred T cell clones persist in vivo in response to low-dose IL-2, preferentially localize to tumor sites and mediate an antigen-specific immune response characterized by the elimination of antigen-positive tumor cells, regression of individual metastases, and minor, mixed or stable responses in 8 of 10 patients with refractory, metastatic disease for up to 21 mo.

Feasibility of using genetic linkage analysis to identify the genes encoding T cell-defined minor histocompatibility antigens.

We have evaluated the utility of genetic linkage analysis to identify genes that encode minor histocompatibility antigens using vaccinia virus vectors as a simple and convenient method for transient expression of class I MHC molecules in lymphoblastoid cell lines. As a test case, we used a CTL clone that recognizes HA-8, a minor histocompatibility antigen encoded by the KIAA0020 gene and presented by HLA-A*0201. EBV-transformed B cell lines from individuals in three large pedigrees from the CEPH reference family collection were infected with a recombinant vaccinia virus vector encoding an HLA-A*0201 transgene, which led to high level expression of the MHC restricting allele HLA-A*0201 on the cell surface. HA-8 expression in the vaccinia-infected target cells was then determined using standard in vitro cytotoxicity assays. Pairwise linkage analysis of the segregation of HA-8 expression in these pedigrees demonstrated that the HA-8 gene was tightly linked with a cluster of marker loci located on the distal portion of chromosome 9p. Analysis of 9p marker haplotypes for individuals in the three families identified several individuals with recombinant haplotypes, and these recombination events were used to refine the precision of the HA-8 gene localization further. The data collectively indicate that the HA-8 gene is localized to a 10.3 cM (corresponding to 3.9 Mb) interval of distal 9p that is thought to encode at least 11 genes, including KIAA0020. These results demonstrate that linkage analysis can be used to map minor histocompatibility genes with high precision and accuracy. Over the next years, refinement and annotation of the human genome sequence will undoubtedly increase the utility of linkage analysis as a tool for identifying minor histocompatibility antigen genes.

Non-myeloablative transplants for malignant disease.

This article discusses changes in the way hematopoietic stem cell allotransplants may be carried out in the future to treat patients with malignant hematological diseases. Specifically, the focus has shifted away from attempts at eradicating underlying diseases through toxic high-dose chemoradiation therapy towards using the stem cell donor's immune cells for that purpose (allogeneic graft-versus-tumor effect). The non-myeloablative transplant approaches hold promise in reducing the morbidity and mortality associated with conventional high-dose chemoradiation therapy, and they allow allogeneic transplants in elderly or medically infirm patients who are at present not candidates for transplantation. In the future, specific graft-versus-tumor responses may become possible by eliciting donor T cell responses to tumor-associated minor histocompatibility antigens. In Section I, Dr. Rainer Storb describes experimental studies in random-bred dogs that rely on non-cytotoxic immunosuppressive agents to establish stable allografts. Powerful postgrafting immunosuppression, traditionally directed at preventing graft-versus-host disease (GVHD), is also used to overcome host-versus-graft (HVG) reactions, thereby dramatically reducing the need for intensive immunosuppressive conditioning programs. Preclinical canine studies have been translated into the clinical setting for treatment of elderly or medically infirm patients with malignant hematological diseases. The pretransplant conditioning has been reduced to a single dose of 2 Gy total body irradiation (TBI) with or without fludarabine. The lack of toxicity makes it possible for transplants to be conducted in the outpatient setting. Multicenter trials have been initiated, and more than 300 patients have been successfully treated with hematopoietic stem cell grafts both from related and unrelated HLA-matched donors. In Section II, Dr. Richard Champlin describes clinical studies with therapeutic strategies that utilize relatively non-toxic, nonmyeloablative disease-specific preparative regimens incorporating fludarabine, together with other chemotherapeutic agents, to achieve disease suppression and engraftment of allogeneic hematopoietic cells and to allow subsequent infusions of donor lymphocytes. Remissions have been seen in patients with acute myelocytic, chronic myelocytic, chronic lymphocytic, leukemias, lymphomas, and myelomas. In Section III, Dr. Stanley Riddell and colleagues describe studies on isolation of T cells reactive with minor histocompatibility (H) antigens and involved both in GVHD and graft-versus-leukemia (GVL) responses. For example, the gene encoding a novel H-Y antigen in humans has been identified and shown to exhibit restricted tissue expression. Acute myelocytic leukemia stem cells were demonstrated to express the H-Y antigen and additional minor H antigens, and engraftment of such cells in NOD/SCID mice could be selectively prevented by minor antigen-specific T-cell clones. An autosomal encoded human minor H antigen associated with chronic GVHD has been demonstrated. A trial evaluating therapy of relapsed acute myelocytic leukemia or acute lymphoblastic leukemia after allogeneic stem cell transplantation with T-cell clones specific for recipient minor H antigens has been initiated.

Targeting antigen in mature dendritic cells for simultaneous stimulation of CD4+ and CD8+ T cells.

Due to their potent immunostimulatory capacity, dendritic cells (DC) have become the centerpiece of many vaccine regimens. Immature DC (DCimm) capture, process, and present Ags to CD4(+) lymphocytes, which reciprocally activate DCimm through CD40, and the resulting mature DC (DCmat) loose phagocytic capacity, but acquire the ability to efficiently stimulate CD8(+) lymphocytes. Recombinant vaccinia viruses (rVV) provide a rapid, easy, and efficient method to introduce Ags into DC, but we observed that rVV infection of DCimm results in blockade of DC maturation in response to all activation signals, including CD40L, monocyte-conditioned medium, LPS, TNF-alpha, and poly(I:C), and failure to induce a CD8(+) response. By contrast, DCmat can be infected with rVV and induce a CD8(+) response, but, having lost phagocytic activity, fail to process the Ag via the exogenous class II pathway. To overcome these limitations, we used the CMV protein pp65 as a model Ag and designed a gene containing the lysosomal-associated membrane protein 1 targeting sequence (Sig-pp65-LAMP1) to target pp65 to the class II compartment. DCmat infected with rVV-Sig-pp65-LAMP1 induced proliferation of pp65-specific CD4(+) clones and efficiently induced a pp65-specific CD4(+) response, suggesting that after DC maturation the intracellular processing machinery for class II remains intact for at least 16 h. Moreover, infection of DCmat with rVV-Sig-pp65-LAMP1 resulted in at least equivalent presentation to CD8(+) cells as infection with rVV-pp65. These results demonstrate that despite rVV interference with DCimm maturation, a single targeting vector can deliver Ags to DCmat for the effective simultaneous stimulation of both CD4(+) and CD8(+) cells.

In vivo tracking of tumor-specific T cells.

Novel immunologic assays now enable visualization of the antigen-specific response to an extent not previously possible. Assessment of not only the numeric frequency but also the functional properties of individual tumor-specific T cells in the endogenous and manipulated immune response has provided insights that will facilitate the development of immunotherapeutic strategies.

Costimulation of CD8alphabeta T cells by NKG2D via engagement by MIC induced on virus-infected cells.

NKG2D is an activating receptor that stimulates innate immune responses by natural killer cells upon engagement by MIC ligands, which are induced by cellular stress. Because NKG2D is also present on most CD8alphabeta T cells, it may modulate antigen-specific T cell responses, depending on whether MIC molecules--distant homologs of major histocompatibility complex (MHC) class I with no function in antigen presentation--are induced on the surface of pathogen-infected cells. We found that infection by cytomegalovirus (CMV) resulted in substantial increases in MIC on cultured fibroblast and endothelial cells and was associated with induced MIC expression in interstitial pneumonia. MIC engagement of NKG2D potently augmented T cell antigen receptor (TCR)-dependent cytolytic and cytokine responses by CMV-specific CD28- CD8alphabeta T cells. This function overcame viral interference with MHC class I antigen presentation. Combined triggering of TCR-CD3 complexes and NKG2D induced interleukin 2 production and T cell proliferation. Thus NKG2D functioned as a costimulatory receptor that can substitute for CD28.

The immunogenicity of a new human minor histocompatibility antigen results from differential antigen processing.

Minor histocompatibility antigens (mHAgs) present a significant impediment to organ and bone marrow transplantation between HLA-identical donor and recipient pairs. Here we report the identification of a new HLA-A*0201-restricted mHAg, HA-8. Designation of this mHAg as HA-8 is based on the nomenclature of Goulmy (Goulmy, E. 1996. Curr. Opin. Immunol. 8:75-81). This peptide, RTLDKVLEV, is derived from KIAA0020, a gene of unknown function located on chromosome 9. Polymorphic alleles of KIAA0020 encode the alternative sequences PTLDKVLEV and PTLDKVLEL. Genotypic analysis demonstrated that the HA-8-specific cytotoxic T lymphocyte (CTL) clone SKH-13 recognized only cells that expressed the allele encoding R at P1. However, when PTLDKVLEV was pulsed onto cells, or when a minigene encoding this sequence was used to artificially translocate this peptide into the endoplasmic reticulum, it was recognized by CTLs nearly as well as RTLDKVLEV. This indicates that the failure of CTLs to recognize cells expressing the PTLDKVLEV-encoding allele of KIAA0020 is due to a failure of this peptide to be appropriately proteolyzed or transported. Consistent with the latter possibility, PTLDKVLEV and its longer precursors were transported poorly compared with RTLDKVLEV by transporter associated with antigen processing (TAP). These studies identify a new human mHAg and provide the first evidence that minor histocompatibility differences can result from the altered processing of potential antigens rather than differences in interaction with the relevant major histocompatibility complex molecule or T cell receptor.