Establishment and Characterization of a Cell Based Artificial Antigen-Presenting Cell for Expansion and Activation of CD8+ T Cells Ex Vivo

Abstract Adoptive transfer of ex vivo expanded CMV-specific T cells is an effective approach, and an attractive alternative to using anti-virals to manage CMV infection for HSCT recipients. We recently published a robust approach to expanding CMV-specific CTL based on infection of autologous EBV-LCL with the attenuated poxvirus, Modified Vaccinia Ankara (MVA), expressing CMV pp65, pp150, and IE1 proteins. This approach causes vigorous, up to 500fold expansions in as little as 12–14 days of memory CD8+ T cells specific for these immunodominant antigens. In order to improve the specificity of the expanded T cells, a method was sought to derive effective antigen presenting cells (APC) that avoided the use of EBV-LCL. Of equal importance is to develop an expansion approach that avoids the need to involve virally infected APC in developing a clinical product. Our preliminary observation is that rMVA can infect PBMC in vitro, causing high levels of expression of recombinant CMV antigens. To be permissible for high level expression from rMVA, fresh PBMC were treated with different combinations of single-stranded CpG-containing phosphorothioate backbone oligonucleotides (ODN). A three-day incubation with a combination of two ODN (ODN # 2006 and 2216) which are known to stimulate both plasmacytoid dendritic and B-cells were found to reproducibly generate a highly rMVA infectable population of PBMC. In all five healthy CMV-positive donors tested, CpG ODN treated autologous PBMC, infected with recombinant rMVA, elicited a 20-fold average expansion of CMV-specific CD8+ T cells, in 10 days. Several different rMVA expressing CMV genes were evaluated, including a novel vector expressing the UL44 gene product, an immunodominant target of the host cellular immune response. The expanded T cell populations showed minimal alloreactivity, and exhibited high levels of CMV-specific MHC Class I tetramer binding, epitope-specific cytokine production, and cytotoxic activity. The availability of a source of autologous professional APC that can be used after only 3 days of priming, enhances the attractiveness of using rMVA for adoptive immunotherapy for HSCT recipients or donor vaccination.

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Artificial Antigen Presenting Cell Can Be Used To Propagate Genetically Modified CD19-Specific T Cells through Chimeric Antigen Receptor.

Blood ◽

10.1182/blood.v106.11.1295.1295 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1295-1295

Author(s):

Tontanai Numbenjapon ◽

Lisa Marie A. Serrano ◽

Simon Olivares ◽

Wen-Chung Chang ◽

Harjeet Singh ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Chimeric Antigen Receptor ◽

Cell Activation ◽

Genetically Modified ◽

K562 Cells ◽

Antigen Presenting Cell ◽

Artificial Antigen ◽

Antigen Presenting

Abstract The safety and feasibility of adoptive immunotherapy, using CD19-specific T cells that have been genetically modified to express a chimeric antigen receptor (CAR) and numerically expanded ex vivo, need to be addressed. Second-generation trials are being developed incorporating improvements into the design of the CAR as well as the manufacturing processes. Here we describe a platform for propagating CD19-specific T cells through an artificial antigen presenting cell (aAPC) which co-expresses CD19 and T-cell co-stimulatory molecules to provide a fully-competent T-cell activation signal leading to T-cell proliferation. K562 cells were selected as the platform for the aAPCs since (i) they have previously been used in compliance with current good manufacturing practice (cGMP), (ii) they express the desired endogenous T-cell adhesion molecules, and (iii) they fail to express classical HLA class I/II molecules and thus are not targets for a T-cell mediated allogeneic immune response. Therefore, K562 cells were genetically modified to co-express CD19 and both of the T-cell co-stimulatory molecules 4-1BBL (CD137L) and MICA. We then tested the ability of these K562 aAPCs to expand T cells expressing a new CD19-specific CAR designated CD19RCD28. This CAR utilizes a CD19-specific scFv to bind to CD19 independent of MHC and confers an activation signal to genetically modified T cells through both CD28 and CD3-ζ. The CD19RCD28+ T cells could be rapidly expanded (50-fold in 14 days) when cultured in the presence of recombinant human IL-2 and irradiated K562 aAPCs (1:50 ratio, T cell to aAPC). The use of freshly thawed aAPCs improved the practicality of using this antigen-driven expansion method in compliance with cGMP. The numerical expansion of the genetically modified T cells was associated with an increased CAR cell-surface expression, from 17 ± 11% (mean ± SD) before co-culture compared with 44 ± 8% (mean ± SD) after co-culture with the aAPCs, which is consistent with T-cell activation through the CAR. A 3H-thymidine incorporation assay was used to demonstrate that CD19 on the K562 aAPC was necessary, but not sufficient, to proliferate CD19RCD28+ T cells. Furthermore, this proliferation assay demonstrated that co-expression of both 4-1BBL (CD137L) and MICA along with CD19 resulted in the most efficient proliferation of the genetically modified T cells. The propagation of CAR+ T cells on antigen+ aAPCs may thus (i) avoid the need for allogeneic peripheral blood mononuclear feeder cells, which are expensive and time-consuming to prepare in compliance with cGMP, (ii) select in vitro for genetically modified T cells with proven CAR-dependent replicative capacity, and (iii) provide conditions for the outgrowth of subpopulations of T cells within a bulk culture that have increased transgene expression. The feasibility of this new T-cell propagation method using aAPC will be tested in the upcoming clinical trials.

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Large Scale Ex Vivo Expansion of Γδ T Cells Using Artificial Antigen Presenting Cells for the Treatment of Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2020-136174 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 7-8

Author(s):

Justin C. Boucher ◽

Bin Yu ◽

Gongbo Li ◽

Bishwas Shrestha ◽

Jeffrey E. Lancet ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Research Funding ◽

Large Scale ◽

Ex Vivo ◽

Γδ T Cells ◽

Artificial Antigen ◽

Antigen Presenting ◽

Acute Myeloid

Patients with relapsed or refractory acute myeloid leukemia (AML) are at increased risk of mortality. Higher γδ T cell count in a bone marrow or peripheral blood of patients with leukemia is associated with better survival. However, γδ T cells are rare in the blood and functionally impaired or exhausted in patients with malignancies. Promising results are reported on the treatment of various malignancies with in vivo expansion of autologous γδ T cells using zoledronic acid (zol) and IL-2. Here we demonstrated that zol and IL-2, in combination with a novel genetically engineered K562 CD3/CD137L/CD28/IL15RA quadruplet artificial antigen presenting cell (aAPC), efficiently expand allogeneic donor-derived γδ T cells using a GMP-compliant protocol sufficient to achieve cell doses for future clinical use. We achieved a 633-fold expansion of γδ T cells after day 10 of co-culture with aAPC, the majority of which exhibited central (47%) and effector (43%) memory phenotypes. Additionally, >90% of the expanded γδ T cells expressed NKG2D, while they have low cell surface expression of PD1 and LAG2 inhibitory checkpoint receptors. In vitro real-time cytotoxicity analysis showed that expanded, previously cryopreserved, γδ T cells were effective in killing target cells. Our results demonstrate that large scale ex vivo expansion of donor-derived γδ T cells can be achieved with the use of CD3/CD137L/CD28/IL15RA quadruplet aAPC and zol/IL-2 for clinical application as promising antineoplastic immunotherapy. Figure 1 Disclosures Lancet: Abbvie: Consultancy; Agios Pharmaceuticals: Consultancy, Honoraria; Astellas Pharma: Consultancy; Celgene: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; ElevateBio Management: Consultancy; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy. Sallman:Celgene, Jazz Pharma: Research Funding; Agios, Bristol Myers Squibb, Celyad Oncology, Incyte, Intellia Therapeutics, Kite Pharma, Novartis, Syndax: Consultancy. Bejanyan:Kiadis Pharma: Membership on an entity's Board of Directors or advisory committees.

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Artificial Antigen Presenting Cells Expand NY-ESO-1 Antigen-Specific CD8+ T Cells From Patients with Melanoma

Blood ◽

10.1182/blood.v118.21.4309.4309 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4309-4309

Author(s):

Marcela V. Maus ◽

Isabelle Riviere ◽

Oriana Borquez-Ojeda ◽

Xiuyan Wang ◽

Jianda Yuan ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Conflicts Of Interest ◽

Adoptive Cell Therapy ◽

Scale Up ◽

Antigen Presenting Cells ◽

Synovial Cell ◽

Melanoma Tumor ◽

Artificial Antigen ◽

Antigen Presenting

Abstract Abstract 4309 The NY-ESO-1 antigen is an attractive prototype cancer-testis (CT) antigen because it is expressed in a wide variety of tumors, including myeloma, melanoma, and synovial cell sarcoma, among others, but has restricted expression in normal tissues (testis and placenta). Based on our previous experience with 3T3-based artificial antigen presenting cells (AAPCs) in expanding influenza and CMV-specific T cells, we hypothesized that AAPCs expressing the NY-ESO-1 antigen and optimal costimulatory molecules would efficiently expand NY-ESO-1 specific T cells for potential use as adoptive immunotherapy. Accordingly, AAPCs were transduced to express the common MHC allele HLA-A*0201, beta2-microglobulin, and the full-length NY-ESO-1 protein, together with the T cell costimulatory ligands CD58, CD54, and CD80. These A2/NY-ESO-1 AAPCs expanded NY-ESO-1 specific T cells from normal donors after prolonged culture (4 weeks). In patients with melanoma who had detectable NY-ESO-1 antibodies, AAPC stimulation of PBMC expanded NY-ESO-1 antigen-specific CD8+ T cells approximately 3–4 logs in 3 weeks. These expanded T cells were functional in that they lysed peptide-pulsed targets (T2 cells) and native melanoma tumor targets (SK Mel 37). Expanded CD8+ T cells also produced interferon-gamma, MIP-1 beta, TNF-alpha and CD107a (but not IL-2) in response to antigen, indicating a polyfunctional response. We are currently performing scale-up experiments and pre-clinical validations of these AAPCs to embark on a clinical trial of adoptive cell therapy of NY-ESO1-specific T cells in patients with NY-ESO1-expressing tumors. Disclosures: No relevant conflicts of interest to declare.

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