Enrichment and Expansion with Nanoscale Artificial Antigen Presenting Cells for Adoptive Immunotherapy

Abstract Abstract 4086 Poster Board III-1021 We have recently established a panel of artificial antigen-presenting cells (AAPCs) each expressing a single HLA class I allele (such as A0201, A0301, A2402, B0702, C0401) and costimulatory molecules (B7.1, ICAMI, LFA3, b2-microglobulin). These cells were capable of eliciting T cells responses specific for immunogenic epitopes of the cytomegalovirus protein CMVpp65 presented by single HLA allele expressed by each AAPC (J.Immunol, 2009;183(4):2837-50). The initial studies with HLA-A0201 expressing AAPCs had suggested that they could sensitize the T cells against single immunogenic A0201-binding peptides derived from antigens such as S100 and telomerase. However, there are no data regarding the potentials of AAPCs expressing other HLA alleles to present epitopes of endogenous self antigens and stimulate specific T cells. We have recently defined a series of immunogenic epitopes for WT1 that can be presented by each of the HLA alleles represented in this panel, by an epitope mapping analysis of WT1 specific T cells sensitized in vitro with autologous dendritic cells(DC) loaded with the pool of overlapping 15-mer peptides spanning the sequence of WT1. In the present study we asked whether the established panel of AAPCs expressing different HLA alleles when loaded with the human WT1 peptide pool, could be used to generate T cells directed against this self antigen, whether the peptides recognized by these T cells would be identical to the peptides defined to be immunogenic when presented in the context of the same HLA alleles after stimulation with the autologous DC loaded with the same WT1 pool and further whether AAPCs could also be used to elicit responses against subdominant epitopes presented by an HLA allele expressed on AAPCs that were not elicited among T cells sensitized with peptide pool loaded autologous DC. The pool of 141 synthetic pentadecapeptides each overlapping the next by 11aa loaded on the autologous DC elicited responses in 80% of 14 normal individuals tested. Epitope mapping permitted identification immunodominant WT1 peptide sequences eliciting responses and their restricting HLA allele(s) as determined in a Cr51 release assay against the panel of WT1 peptide loaded EBV transformed B cells matching one of the HLA alleles of the T cell genotype.. AAPCs expressing single HLA allele shared by the same T cells and loaded with the same WT1 total peptide pool elicited responses in each normal individual. In each case the T cells recognized one of the same epitopes defined to be previously immunogenic in man when presented in the context of the HLA allele expressed by the AAPCs. At the same time, single peptide epitopes that were defined to be presented by, for example, HLA A0201 alleles after stimulation with WT1 total pool loaded DCs did not elicit WT1 specific T cells responses if they were loaded on AAPCs expressing another HLA allele such as B0702. In comparisons of T cells sensitized with autologous DCs versus AAPCs loaded with WT1 peptides pool we also found that while AAPCs expressing the HLA allele presenting the immunodominant epitope recognized by T cells sensitized with pool loaded autologous DC also elicited strong responses, AAPCs expressing HLA alleles shared by the responding T cells could also elicit responses against immunogenic but subdominant epitopes that were not generated after sensitization of the same T cells with WT1 peptide pool loaded autologous DCs. Thus, these studies suggest that the panel of murine derived AAPCs can effectively present self antigens, such as WT1, and permits in vitro sensitization and propagation of tumor reactive WT1 peptide specific T cells of desired HLA restriction for the adoptive immunotherapy of WT1+ malignancies. Disclosures: No relevant conflicts of interest to declare.

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Artificial Antigen-Presenting Cells for Use in Adoptive Immunotherapy

The Cancer Journal ◽

10.1097/ppo.0b013e3181eb33a6 ◽

2010 ◽

Vol 16 (4) ◽

pp. 374-381 ◽

Cited By ~ 56

Author(s):

Cameron J. Turtle ◽

Stanley R. Riddell

Keyword(s):

Adoptive Immunotherapy ◽

Antigen Presenting Cells ◽

Artificial Antigen ◽

Antigen Presenting

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Generation of CMV-Specific T-Lymphocytes for Adoptive Immunotherapy: A Comparison of Artificial Antigen-Presenting Cells and Autologous Dendritic Cells Pulsed with CMV-pp65 Protein-Spanning Pentadecapeptide Pools.

Blood ◽

10.1182/blood.v106.11.5546.5546 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5546-5546

Author(s):

Wouter J.W. Kollen ◽

Deepa Trivedi ◽

Matthias Stephan ◽

Michel Sadelain ◽

Richard J. O’Reilly

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Adoptive Immunotherapy ◽

Antigen Presenting Cells ◽

Hla Class I ◽

Treatment And Prevention ◽

Endogenous Protein ◽

Artificial Antigen ◽

Time Required ◽

Antigen Presenting

Abstract Adoptive transfer of virus-specific T-cells constitutes a promising approach for the treatment and prevention of cytomegalovirus (CMV) infections complicating allogeneic HSC transplants. Current strategies employing peptide-pulsed donor-derived dendritic cells (DCs) for antigen presentation are effective, but limited by availability of adequate numbers of DCs and the time required for their generation. We established a series of immediately accessible, replenishable, artificial antigen-presenting cells (AAPCs), consisting of mouse 3T3 cells transduced to express human B7.1, LFA-3, ICAM-1, β2-microglobulin and one HLA class I allele (Papanicolaou et al., Blood 2003). We then compared yields of CMV-peptide-specific T-cells when T-cells from HLA A0201 seropositive donors were sensitized with autologous DCs or HLA A0201 AAPCs loaded with a known HLA A0201-binding immunogenic nonamer of CMV-pp65 (NLVPMVATV) or a pool of pentadecapeptides spanning CMV-pp65, which included the 15-mer No. 123 (AGILARNLVPMVATV) containing this epitope. Peptide loading was performed in the presence or absence of serum in order to distinguish peptide editing mediated by ectopeptidases in the serum or expressed by the different APCs. Results from repeated experiments employing three HLA A0201 CMV-seropositive donors demonstrated that the yields of epitope-specific T-cells following 14–28 day sensitization with AAPCs loaded with the NLVPMVATV nonamer consistently exceeded those generated in response to peptide-loaded autologous DCs, as assessed by enumerating peptide-HLA A0201 tetramer-binding and peptide-specific IFN-γ producing T cells. High and selective yields of NLV-specific T-cells were obtained when the T-cells were sensitized with AAPCs or DCs loaded with the CMV-pp65-spanning pool of 15-mers in the presence or absence of serum suggesting that ectopeptidases expressed on the DCs and AAPCs appropriately clip and edit the 15-mers after binding to HLA A0201. Even higher yields were achieved when the T-cells were sensitized with AAPCs transduced to express the full length CMV-pp65 protein, confirming the potential of the mouse-derived AAPCs to process this protein and load immunogenic epitopes on HLA A0201. T-cells generated in response to peptide-loaded or transduced AAPCs also specifically lysed nonamer-loaded HLA A0201 expressing targets. Taken together, these studies indicate that mouse-derived AAPCs can efficiently process endogenous protein and edit exogenous 15-mer peptides for binding and presentation by the single expressed HLA allele. Use of these AAPCs loaded with overlapping peptides or transduced to express an immunogenic protein may thus provide advantages in terms of immunogenicity, immediate accessibility and broad applicability for rapid production of antigen-specific T-cells for adoptive immunotherapy.

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Artificial Antigen-Presenting Cells Permit Selective In Vitro Generation of CMV-Specific T-Cells of Desired HLA Allelic Restriction for Adoptive Immunotherapy in Recipients of HLA Disparate Allografts.

Blood ◽

10.1182/blood.v106.11.1298.1298 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1298-1298 ◽

Cited By ~ 1

Author(s):

Deepa Trivedi ◽

Wouter J.W. Kollen ◽

Lorna Barnett ◽

Michel Sadelain ◽

Richard J. O’Reilly

Keyword(s):

T Cells ◽

T Cell ◽

Adoptive Immunotherapy ◽

Peptide Pool ◽

Antigen Presenting Cells ◽

Hla Alleles ◽

Hla Restriction ◽

Artificial Antigen ◽

Antigen Presenting

Abstract Cytomegalovirus (CMV) infections remain a major cause of morbidity and mortality following allogeneic hematopoietic cell transplantation. For recipients of HLA haplotype disparate grafts, the risk of CMV infection is especially high and extends late into the post-transplant period. Early clinical trials indicate that adoptive transfer of ex vivo generated CMV-specific T-cells from the donor can be effective in the treatment and prevention of such infections. However, because seropositive donor T-cells sensitized with autologous infected or antigen loaded APCs regularly exhibit a repertoire of CMV-specific T-cells restricted by one or two immunodominant HLA alleles, such T-cells will only be effective if these immunodominant alleles are shared by the infected host. For example, among donors inheriting HLA-B*0702, CD8+ T-cell responses to CMV-pp65 peptides are usually, and often exclusively, restricted by this allele. To address this limitation, we tested whether artificial antigen-presenting cells, consisting of murine 3T3 cells transduced to express human B7.1, LFA1, ICAM1, β 2M and HLA-A*0201 alpha chain (Papanicolaou et al., Blood 2003), could be used to generate CMV-specific T-cells restricted by this allele in a series of normal donors inheriting HLA-A*A0201 and HLA-B*0702. Accordingly, T-cells were sensitized in vitro with either autologous monocyte-derived DCs or AAPCs expressing HLA-A*A0201, each loaded with a pool of 138 overlapping pentadecapeptides spanning the CMV-pp65 protein. Thereafter, specificity of responding T-cells was identified by mapping epitopes using an intersecting matrix of peptide subpools and measuring T-cells producing IFN-γ following secondary restimulation. HLA restriction was identified by analysis of T-cell cytotoxic responses against panels of EBV BLCL sharing single HLA alleles with the donor and loaded with the targeted peptide. Findings were confirmed by quantitation of T-cell binding tetramers containing targeted epitopes bound to either HLA-A*0201 or B*0702. In each of the donors, sensitization with DCs loaded with the peptide pool resulted in the generation of CMV-pp65-specific T-cells specific for epitopes predominantly or exclusively presented by HLA-B*0702. In contrast, sensitization with peptide pool loaded AAPC led to the generation of high numbers of T-cells recognizing separate CMV-pp65 epitopes in the context of HLA-A*A0201. These HLA-A*0201-restricted T-cells were also able to lyse peptide-loaded BLCLs or PBMCs expressing HLA-A*0201. Taken together, these studies demonstrate that AAPCs expressing single HLA alleles can be used to generate fully functional T-cells specific for epitopes presented by subdominant HLA alleles. This strategy may thus permit generation of virus-specific T-cells of desired HLA-restriction for adoptive immunotherapy in HLA-disparate transplant recipients.

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