Immunotherapy Targets in Pediatric Cancer

Immunotherapy for cancer has shown increasing success and there is ample evidence to expect that progress gleaned in immune targeting of adult cancers can be translated to pediatric oncology. This manuscript reviews principles that guide selection of targets for immunotherapy of cancer, emphasizing the similarities and distinctions between oncogene-inhibition targets and immune targets. It follows with a detailed review of molecules expressed by pediatric tumors that are already under study as immune targets or are good candidates for future studies of immune targeting. Distinctions are made between cell surface antigens that can be targeted in an MHC independent manner using antibodies, antibody derivatives, or chimeric antigen receptors versus intracellular antigens which must be targeted with MHC restricted T cell therapies. Among the most advanced immune targets for childhood cancer are CD19 and CD22 on hematologic malignancies, GD2 on solid tumors, and NY-ESO-1 expressed by a majority of synovial sarcomas, but several other molecules reviewed here also have properties which suggest that they too could serve as effective targets for immunotherapy of childhood cancer

PII: S1471-4906(01)02028-2

Recent evidence has implicated interleukin-7 (IL-7) as a master regulator of T-cell homeostasis, based upon its essential role in the homeostatic expansion of naive T-cell populations in response to low-affinity antigens (Ags) and its capacity to enhance dramatically the expansion of peripheral T-cell populations in response to high-affinity Ags. Furthermore, T-cell-depleted humans have a unique inverse relationship between the peripheral CD4 + + T-cell count and the level of circulating IL-7. Together, these data suggest that increased amounts of IL-7 become available following T-cell depletion, thus, enhancing the high-and low-affinity Ag-driven expansion of the population of residual, mature T cells and boosting thymic regenerative capacity, as a means to restore T-cell homeostasis

Molecular Alterations in Pediatric Sarcomas: Potential Targets for Immunotherapy

Purpose/results/discussion. Recurrent chromosomal translocations are common features of many human malignancies. While such translocations often serve as diagnostic markers, molecular analysis of these breakpoint regions and the characterization of the affected genes is leading to a greater understanding of the causal role such translocations play in malignant transformation. A common theme that is emerging from the study of tumor-associated translocations is the generation of chimeric genes that, when expressed, frequently retain many of the functional properties of the wild-type genes from which they originated. Sarcomas, in particular, harbor chimeric genes that are often derived from transcription factors, suggesting that the resulting chimeric transcription factors contribute to tumorigenesis. The tumor-specific expression of the fusion proteins make them likely candidates for tumor-associated antigens (TAA) and are thus of interest in the development of new therapies. The focus of this review will be on the translocation events associated with Ewing's sarcomas/PNETs (ES), alveolar rhabdomyosarcoma (ARMS), malignant melanoma of soft parts (MMSP) (clear cell sarcoma), desmoplastic small round cell tumor (DSRCT), synovial sarcoma (SS), and liposarcoma (LS), and the potential for targeting the resulting chimeric proteins in novel immunotherapies

Removal Of Myeloid Cells From Autologous Leukocytes Used For Chimeric Antigen Receptor (Car) T Cell Manufacturing Improves Final Product Consistency And Yields

Early phase clinic trails of T cells genetically engineered to express Chimeric Antigen Receptors (CAR) have been promising. CD19-CAR T cells have been used successfully in a number of clinical trials to treat non-Hodgkin’s lymphoma and acute lymphocytic leukemia (ALL) and clinical trials of GD2-CAR T cells for the treatment of osteosarcoma and neuroblastoma are underway. Most CAR T cell manufacturing protocols make use of autologous peripheral blood mononuclear cell (PBMC) concentrates collected by apheresis, however, the lymphocyte-rich PBMC concentrates are also enriched for monocytes and contain small but variable quantities of red blood cells, platelets and neutrophils and prior to beginning CAR T cell manufacturing the PBMC concentrates are generally enriched for lymphocytes or CD3+ cells. We initially manufactured CD19- and GD2-CAR T cells using autologous PBMC concentrates enriched for T cells by selection with the anti-CD3/CD28 beads. These same anti-CD3/CD28 beads were used to stimulated T cell expansion. While the method was, in general, effective, we found that the quantities of GD2-CAR T cells produced were less than the quantities of CD19 CAR T cells produced. In addition, T cells from some patients failed to expand at all. Further investigation found that the presence of large quantities of monocytes or granulocytes in some PBMC concentrates which was associated with poor in vitro CAR T cell expansion. Myeloid derived suppressor cells (MDSCs) that inhibit T cell proliferation are present in sarcoma and ALL patients. These MDSCs may have a monocyte or neutrophil phenotype. We hypothesized that MDSCs in the PBSC concentrates bound non-specifically to the anti-CD3/CD28 beads and more rigorous enrichment of the starting material for lymphocytes would improve CAR T cell yields and reduce the incidence of manufacturing failures. We modified the T cell enrichment method to include a monocyte-depleting plastic adherence step. This change improved T cell expansion, but it was not completely effective at removing contaminating monocytes and granulocytes and did not completely eliminate manufacturing failures. To provide better depletion of monocytes and granulocytes we subjected PBMC concentrates to counter flow elutriation instrument. We manufactured 8 CD19- and 5 GD2-CAR T cell products from elutriated lymphocytes. All 13 CAR T cell manufacturing procedures yielded sufficient quantities of T cells to meet the dose criteria. The 13 CAR T cell products contained 2,166±1,113 x106 CD3+ cells and 1,064±877 x106 transduced CD3+ T cells. The CAR T cell products manufactured from elutriated lymphocytes yielded significantly more CD3+ cells and transduced CD3+ cells than that of those manufactured from anti-CD3/CD28 bead selected and plastic adherence selected cells. These results show CAR T cell manufacturing yields are greater and more consistent when manufacturing is initiated with lymphocytes that have few contaminating myeloid cells

Identification of Cell Surface Proteins as Potential Immunotherapy Targets in 12 Pediatric Cancers

Technological advances now allow us to rapidly produce CARs and other antibody-derived therapeutics targeting cell surface receptors. To maximize the potential of these new technologies, relevant extracellular targets must be identified. The Pediatric Oncology Branch of the NCI curates a freely accessible database of gene expression data for both pediatric cancers and normal tissues, through which we have defined discrete sets of over-expressed transcripts in 12 pediatric cancer subtypes as compared to normal tissues. We coupled gene expression profiles to current annotation databases (i.e., Affymetrix, Gene Ontology, Entrez Gene), in order to categorize transcripts by their sub-cellular location. In this manner we generated a list of potential immune targets expressed on the cell surface, ranked by their difference from normal tissue. Global differences from normal between each of the pediatric tumor types studied varied, indicating that some malignancies expressed transcript sets that were more highly diverged from normal tissues than others. The validity of our approach is seen by our findings for pre-B cell ALL, where targets currently in clinical trials were top-ranked hits (CD19, CD22). For some cancers, reagents already in development could potentially be applied to a new disease class, as exemplified by CD30 expression on sarcomas. Moreover, several potential new targets shared among several pediatric solid tumors are herein identified, such as MCAM (MUC18), metadherin (MTDH), and glypican-2 (GPC2). These targets have been identified at the mRNA level and are yet to be validated at the protein level. The safety of targeting these antigens has yet to be demonstrated and therefore the identified transcripts should be considered preliminary candidates for new CAR and therapeutic antibody targets. Prospective candidate targets will be evaluated by proteomic analysis including Westerns and immunohistochemistry of normal and tumor tissues

A Role for Thymic Stromal Lymphopoietin in CD4+ T Cell Development

Thymic stromal lymphopoietin (TSLP) signals via a receptor comprising the interleukin (IL)-7 receptor α chain and a distinctive subunit, TSLP receptor (TSLPR), which is most related to the common cytokine receptor γ chain, γc. We have generated TSLPR knockout (KO) mice and found that although these mice had normal lymphocyte numbers, γc/TSLPR double KO mice had a greater lymphoid defect than γc KO mice. This indicates that TSLP contributes to lymphoid development and accounts for some of the residual lymphoid development in γc KO mice and presumably in patients with X-linked severe combined immunodeficiency. Injection of TSLP into γc KO mice induced the expansion of T and B cells. Moreover, sublethally irradiated TSLPR KO mice showed weaker recovery of lymphocyte populations than wild-type (WT) littermates, even when neutralizing anti–IL-7 antibodies were injected. Interestingly, TSLP preferentially stimulated the proliferation and survival of CD4+ single positive thymocytes and peripheral T cells in vitro. Additionally, CD4+ T cells from TSLPR KO mice expanded less efficiently than WT CD4+ T cells in irradiated hosts, and TSLP preferentially expanded CD4+ T cells both in vitro and in vivo. Thus, as compared with other known cytokines, TSLP is distinctive in exhibiting a lineage preference for the expansion and survival of CD4+ T cells

Robust Selections of Various Hematopoietic Cell Fractions on the CliniMACS Plus Instrument

Cell separation technologies play a vital role in the graft engineering of hematopoietic cellular fractions, particularly with the rapid expansion of the field of cellular therapeutics. The CliniMACS Plus Instrument (Miltenyi Biotec) utilizes immunomagnetic techniques to isolate hematopoietic progenitor cells (HPCs), T cells, NK cells, and monocytes. These products are ultimately used for HPC transplantation and for the manufacture of adoptive immunotherapies. We evaluated the viable cell recovery and cell purity of selections and depletions performed on the CliniMACS Plus over a 10-year period at our facility, specifically assessing for the isolation of CD34+, CD4+, CD3+/CD56+, CD4+/CD8+, and CD25+ cells. Additionally, patient- and instrument-related factors affecting these parameters were examined. Viable cell recovery ranged from 32.3 ± 10.2% to 65.4 ± 15.4%, and was the highest for CD34+ selections. Cell purity ranged from 86.3 ± 7.2% to 99.0 ± 1.1%, and was the highest for CD4+ selections. Undesired cell fractions demonstrated a range of 1.2 ± 0.45 to 5.1 ± 0.4 log reductions. Red cell depletions averaged 2.12 ± 0.68 logs, while platelets were reduced by an average of 4.01 ± 1.57 logs. Donor characteristics did not impact viable cell recovery or cell purity for CD34+ or CD4+ cell enrichments; however, these were affected by manufacturing variables, including tubing size, bead quantity, and whether preselection platelet washes were performed. Our data demonstrate the efficient recovery of hematopoietic cellular fractions on the CliniMACS Plus that may be optimized by adjusting manufacturing variables