Targeted drug distribution in tumor extracellular fluid of GD2-expressing neuroblastoma patient-derived xenografts using SN-38-loaded nanoparticles conjugated to the monoclonal antibody 3F8.

Neuroblastoma is a pediatric solid tumor with high expression of the tumor associated antigen disialoganglioside GD2. Despite initial response to induction therapy, nearly 50% of high-risk neuroblastomas recur because of chemoresistance. Here we encapsulated the topoisomerase-I inhibitor SN-38 in polymeric nanoparticles (NPs) surface-decorated with the anti-GD2 mouse mAb 3F8 at a mean density of seven antibody molecules per NP. The accumulation of drug-loaded NPs targeted with 3F8 versus with control antibody was monitored by microdialysis in patient-derived GD2-expressing neuroblastoma xenografts. We showed that the extent of tumor penetration by SN-38 was significantly higher in mice receiving the targeted nano-drug delivery system when compared to non-targeted system or free drug. This selective penetration of the tumor extracellular fluid translated into a strong anti-tumor effect prolonging survival of mice bearing GD2-high neuroblastomas in vivo.

Affinity maturation of T-cell receptor-like antibodies for Wilms tumor 1 peptide greatly enhances therapeutic potential.

WT1126 (RMFPNAPYL) is a human leukocyte antigen-A2 (HLA-A2)-restricted peptide derived from Wilms tumor protein 1 (WT1), which is widely expressed in a broad spectrum of leukemias, lymphomas and solid tumors. A novel T-cell-receptor (TCR)-like single-chain variable fragment (scFv) antibody specific for the T-cell epitope consisting of the WT1/HLA-A2 complex was isolated from a human scFv phage library. This scFv was affinity-matured by mutagenesis combined with yeast display and structurally analyzed using a homology model. This monovalent scFv showed a 100-fold affinity improvement (dissociation constant (KD)=3 nm) and exquisite specificity towards its targeted epitope or HLA-A2(+)/WT1(+) tumor cells. Bivalent scFv-huIgG1-Fc fusion protein demonstrated an even higher avidity (KD=2 pm) binding to the T-cell epitope and to tumor targets and was capable of mediating antibody-dependent cell-mediated cytotoxicity or tumor lysis by chimeric antigen receptor-expressing human T- or NK-92-MI-transfected cells. This antibody demonstrated specific and potent cytotoxicity in vivo towards WT1-positive leukemia xenograft that was HLA-A2 restricted. In summary, T-cell epitopes can provide novel targets for antibody-based therapeutics. By combining phage and yeast displays and scFv-Fc fusion platforms, a strategy for developing high-affinity TCR-like antibodies could be rapidly explored for potential clinical development.

Retargeting NK92 cells using an HLA-A2-restricted, EBNA3C-specific chimeric antigen receptor.

Advances in adoptive cell immunotherapy have led to several promising options for cancer patients. Single-chain variable fragments (scFvs) were isolated from a human phage display library by panning on recombinant human leukocyte antigen (HLA)-A2-peptide complexes. A scFv (EBNA Clone 315) specific for HLA-A2 carrying a 10 amino acid peptide (LLDFVRFMGV) derived from the Epstein-Barr virus latent protein EBNA3C was fully characterized. EBNA Clone 315 displayed exquisite specificity toward its targeted T-cell epitope (TCE) and did not cross-react with the free peptide, HLA-A2 complexes, which carried irrelevant peptides, or HLA-A2(-) cells. Furthermore, after engineering into a scFv-Fc fusion protein, we were able to determine its affinity, detection sensitivity, and ability to induce antibody-dependent cellular cytotoxicity (ADCC). As a proof-of-principle, a chimeric antigen receptor (CAR) version of EBNA Clone 315 was used to reprogram NK92MI cells. CAR-expressing NK92MI cells showed highly specific and potent cytotoxicity toward the targeted TCE, with detection sensitivity of approximately 25 molecules and cytolytic capacity threefold greater than scFv-Fc-mediated ADCC. For the first time, we show the successful reprogramming of non-T cells toward a specific TCE using a CAR.

Topotecan, thiotepa, and carboplatin for neuroblastoma: failure to prevent relapse in the central nervous system.

We report on a three-drug myeloablative regimen designed to consolidate remission and to prevent central nervous system (CNS) relapse of high-risk neuroblastoma (NB). Sixty-six NB patients received topotecan 2 mg/m2/day, x 4 days; thiotepa 300 mg/m2/day, x 3 days; and carboplatin approximately 500 mg/m2/day, x 3 days. Post-SCT treatments included radiotherapy, immunotherapy, 13-cis-retinoic acid, +/-oral etoposide. Significant nonhematologic toxicities were mucositis and skin-related in all patients, convulsions in three patients, and cardiac failure and venocclusive disease of liver in one patient each. Grade 2 hepatotoxicity led to truncating cytoreduction in two patients; both later relapsed in brain. Among 46 patients transplanted in first complete/very good partial remission (CR/VGPR), event-free survival is 54% (s.e.+/-8%) at 36 months post-SCT; notable events were three non-NB-related deaths (adenovirus on day +9, bowel necrosis at 5 months, multiorgan failure at seven months) and four relapses in brain. Of 12 patients transplanted with evidence of NB, two became long-term event-free survivors and two relapsed in the brain. Of eight patients transplanted in second or greater CR/VGPR, one became a long-term event-free survivor and seven relapsed though not in the CNS. This regimen has manageable toxicity but does not prevent CNS relapse.

Comprehensive analysis of the 9p21 region in neuroblastoma suggests a role for genes mapping to 9p21-23 in the biology of favourable stage 4 tumours.

Chromosome 9p21 is frequently deleted in many cancers. Previous reports have indicated that 9p21 LOH is an uncommon finding in neuroblastoma (NB), a tumour of childhood. We have performed an extensive analysis of 9p21 and genes located in this region (cyclin-dependent kinase inhibitor 2A - CDKN2A/p16(INK4a), CDKN2A/p14(ARF), CDKN2B/p15(INK4b), MTAP, interferon alpha and beta cluster). LOH was detected in 16.4% of 177 NB. The SRO was identified between markers D9S1751 and D9S254, at 9p21-23, a region telomeric to the CDKN2A and MTAP genes. A significantly better overall and progression-free survival was detected in stage 4 patients displaying 9p21-23 LOH. Hemizygous deletion of the region harbouring the CDKN2A and CDKN2B loci was identified in two tumours by means of fluorescent in situ hybridisation and MTAP was present by immunostaining in all but one tumour analysed. The transcriptional profile of tumours with 9p21-23 LOH was compared to that of NB displaying normal 9p21-23 status by means of oligonucleotide microarrays. Four of the 363 probe sets downregulated in tumours with 9p21-23 LOH were encoded by genes mapping to 9p22-24. The only well-characterised transcript among them was nuclear factor I-B3. Our results suggest a role for genes located telomeric of 9p21 in good risk NB.

Expression of SSX-2 and SSX-4 genes in neuroblastoma.

The SSX genes are members of the family of cancer/testis antigens that encode tumor-associated antigens recognizable by autologous cytolytic T lymphocytes. Their expression is common in tumors of diverse lineages and absent in normal tissues except testis and thyroid. In this study, sixty-seven neuroblastomas (NB) (12 stage 1, 13 stage 2, 12 stage 3, 12 stage 4S and 13 stage 4) were examined by RT-PCR and a sensitive chemiluminescent detection method for SSX-2 and SSX-4 expression. Seventy-two percent (13/18) of stage 4 NB expressed SSX-2 and 67% (12/18) expressed SSX-4. SSX-2 and SSX-4 positivity correlated with metastatic NB stage 4 (p=0.02 and p=0.006, respectively). Sensitivity experiments showed SSX-2 detection was one tumor cell in 10(6) normal cells, and one in 10(4) for SSX-4. All normal tissues (n=6), with the exception of testis, normal bone marrow (BM, n=12) and normal peripheral blood (PBL, n=10) were negative for SSX-2 and SSX-4 expression. Thirty-two BM and 14 PBL obtained from 35 stage 4 NB patients at 24 months from their diagnosis were evaluated for SSX-2 expression. Unlike another cancer/testis antigen, GAGE, only one BM sample was positive, and no prognostic utility could be established. Further investigation of SSX expression at other relevant time points is warranted.