Delta One T Cells for Immunotherapy of Chronic Lymphocytic Leukemia: Clinical-Grade Expansion/Differentiation and Preclinical Proof of Concept.
OBJECTIVE
The Vδ1+subset of γδ T lymphocytes is a promising candidate for cancer immunotherapy, but the lack of suitable expansion/differentiation methods has precluded therapeutic application. We set out to develop and test (preclinically) a Vδ1+T-cell-based protocol that is good manufacturing practice compatible and devoid of feeder cells for prompt clinical translation.


METHODS
We tested multiple combinations of clinical-grade agonist antibodies and cytokines for their capacity to expand and differentiate (more than 2-3 weeks) Vδ1+T cells from the peripheral blood of healthy donors and patients with chronic lymphocytic leukemia (CLL). We characterized the phenotype and functional potential of the final cellular product, termed Delta One T (DOT) cells, in vitro and in vivo (xenograft models of CLL).


RESULTS
We describe a very robust two-step protocol for the selective expansion (up to 2,000-fold in large clinical-grade cell culture bags) and differentiation of cytotoxic Vδ1+(DOT) cells. These expressed the natural cytotoxicity receptors, NKp30 and NKp44, which synergized with the T-cell receptor to mediate leukemia cell targeting in vitro When transferred in vivo, DOT cells infiltrated tumors and peripheral organs, and persisted until the end of the analysis without showing signs of loss of function; indeed, DOT cells proliferated and produced abundant IFNγ and TNFα, but importantly no IL17, in vivo Critically, DOT cells were capable of inhibiting tumor growth and preventing dissemination in xenograft models of CLL.


CONCLUSIONS
We provide a clinical-grade method and the preclinical proof of principle for application of a new cellular product, DOT cells, in adoptive immunotherapy of CLL. Clin Cancer Res; 22(23); 5795-804. ©2016 AACR.
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal.|Lymphact - Lymphocyte Activation Technologies S.A., Coimbra, Portugal.
Afonso R
AR
Almeida
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal.|Lymphact - Lymphocyte Activation Technologies S.A., Coimbra, Portugal.
Daniel V
DV
Correia
Department of Bioengineering and IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
Ana
A
Fernandes-Platzgummer
Department of Bioengineering and IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
Cláudia L
CL
da Silva
Instituto Português de Oncologia de Lisboa - Francisco Gentil, Lisboa, Portugal.|Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisboa, Portugal.
Maria Gomes
MG
da Silva
Lymphact - Lymphocyte Activation Technologies S.A., Coimbra, Portugal.
Diogo Remechido
DR
Anjos
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal. bssantos@fm.ul.pt.|Lymphact - Lymphocyte Activation Technologies S.A., Coimbra, Portugal.
Bruno
B
Silva-Santos
D000818:Animals; D002454:Cell Differentiation / Q000276:immunology*; D045744:Cell Line, Tumor; D003602:Cytotoxicity, Immunologic / Q000276:immunology; D005260:Female; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods; D007371:Interferon-gamma / Q000276:immunology; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000276:immunology* / Q000628:therapy*; D018655:Lymphocyte Count / Q000379:methods; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000276:immunology*; D016176:T-Lymphocyte Subsets / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology
D016692:Receptors, Antigen, T-Cell, gamma-delta; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma