Combining adoptive cellular and immunocytokine therapies to improve treatment of B-lineage malignancy.

Currently, the lineage-specific cell-surface molecules CD19 and CD20 present on many B-cell malignancies are targets for both antibody- and cell-based therapies. Coupling these two treatment modalities is predicted to improve the antitumor effect, particularly for tumors resistant to single-agent biotherapies. This can be shown using an immunocytokine, composed of a CD20-specific monoclonal antibody fused to biologically active interleukin 2 (IL-2), combined with ex vivo expanded human umbilical cord blood-derived CD8(+) T cells, that have been genetically modified to be CD19 specific, for adoptive transfer after allogeneic hematopoietic stem-cell transplantation. We show that a benefit of targeted delivery of recombinant IL-2 by the immunocytokine to the CD19(+)CD20(+) tumor microenvironment is improved in vivo persistence of the CD19-specific T cells, and this results in an augmented cell-mediated antitumor effect. Phase I trials are under way using anti-CD20-IL-2 immunocytokine and CD19-specific T cells as monotherapies, and our results warrant clinical trials using combination of these two immunotherapies.

Differentiation of naive cord-blood T cells into CD19-specific cytolytic effectors for posttransplantation adoptive immunotherapy.

Disease relapse is a barrier to achieving therapeutic success after unrelated umbilical cord-blood transplantation (UCBT) for B-lineage acute lymphoblastic leukemia (B-ALL). While adoptive transfer of donor-derived tumor-specific T cells is a conceptually attractive approach to eliminating residual disease after allogeneic hematopoietic stem cell transplantation, adoptive immunotherapy after UCBT is constrained by the difficulty of generating antigen-specific T cells from functionally naive umbilical cord-blood (UCB)-derived T cells. Therefore, to generate T cells that recognize B-ALL, we have developed a chimeric immunoreceptor to redirect the specificity of T cells for CD19, a B-lineage antigen, and expressed this transgene in UCB-derived T cells. An ex vivo process, which is compliant with current good manufacturing practice for T-cell trials, has been developed to genetically modify and numerically expand UCB-derived T cells into CD19-specific effector cells. These are capable of CD19-restricted cytokine production and cytolysis in vitro, as well as mediating regression of CD19+ tumor and being selectively eliminated in vivo. Moreover, time-lapse microscopy of the genetically modified T-cell clones revealed an ability to lyse CD19+ tumor cells specifically and repetitively. These data provide the rationale for infusing UCB-derived CD19-specific T cells after UCBT to reduce the incidence of CD19+ B-ALL relapse.

Enhanced antilymphoma efficacy of CD19-redirected influenza MP1-specific CTLs by cotransfer of T cells modified to present influenza MP1.

To enhance the in vivo antitumor activity of adoptively transferred, CD19-specific chimeric antigen receptor (CAR)-redirected cytotoxic T lymphocytes (CTLs), we studied the effect of restimulating CAR(+) CTLs through their endogenous virus-specific T-cell antigen receptor (TcR) by the cotransfer of engineered T-cell antigen-presenting cells (T-APCs). Using influenza A matrix protein 1 (MP1) as a model antigen, we show that ex vivo-expanded CD4(+) and CD8(+) T-APCs expressing a hygromycin phosphotransferase-MP1 fusion protein (HyMP1) process and present MP1 to autologous human leukocyte antigen (HLA)-restricted, MP1-specific CD4(+) and CD8(+) CTL precursors. The MP1-specific CTLs are amenable to subsequent genetic modification to express a CD19-specific CAR, designated CD19R, and acquire HLA-unrestricted reactivity toward CD19(+) leukemia and lymphoma tumor targets while maintaining HLA-restricted MP1 specificity. The restimulation of MP1xCD19 dual-specific CTLs in vivo by the adoptive transfer of irradiated HyMP1(+) T-APCs resulted in the enhanced antilymphoma potency of bispecific effector cells, as measured by elimination of the biophotonic signal of established firefly luciferase-expressing Burkitt lymphoma xenografts in nonobese diabetic/severe combined immunodeficiency (NOD/scid) animals compared with control groups restimulated by Hy(+)MP1(neg) T-APCs. Engineered T-APCs are a novel and versatile antigen-delivery system for generating antigen-specific T cells in vitro and enhancing the in vivo effector functioning of CAR-redirected antitumor effector cells.

T-cell clones can be rendered specific for CD19: toward the selective augmentation of the graft-versus-B-lineage leukemia effect.

Relapse of B-lineage acute lymphoblastic leukemia (B-ALL) after allogeneic hematopoietic stem cell transplantation (HSCT) commonly results from the failure of a graft-versus-leukemia (GVL) effect to eradicate minimal residual disease. Augmenting the GVL effect by the adoptive transfer of donor-derived B-ALL-specific T-cell clones is a conceptually attractive strategy to decrease relapse rates without exacerbating graft-versus-host disease (GVHD). Toward this end, we investigated whether a genetic engineering approach could render CD8(+) cytotoxic T lymphocytes (CTLs) specific for tumor cells that express the B-cell lineage cell surface molecule CD19. This was accomplished by the genetic modification of CTLs to express a chimeric immunoreceptor composed of a CD19-specific single-chain immunoglobulin extracellular targeting domain fused to a CD3-zeta intracellular signaling domain. CD19-redirected CTL clones display potent CD19-specific lytic activity and chimeric immunoreceptor-regulated cytokine production and proliferation. Because B-ALL cells can evade T-cell/natural killer- cell recognition by down-regulation of cell surface accessory molecules that participate in the formation of a functional immunologic synapse, we compared the CD19-specific effector function of genetically modified CD8(+) CTLs toward CD19(+) cells with disparate levels of intercellular adhesion molecule 1 (ICAM-1), leukocyte function-associated antigen 1 (LFA-1), and LFA-3. We observed that recognition of B-lineage tumor lines by CD19-specific CTLs was not impaired by low levels of ICAM-1, LFA-1, and LFA-3 cell surface expression, a functional attribute that is likely a consequence of our high-affinity CD19-specific chimeric immunoreceptor. Furthermore, the CD19-specific CTLs could lyse primary B-ALL blasts. These preclinical observations form the basis for implementing clinical trials using donor-derived CD19-specific T-cell clones to treat or prevent relapse of B-ALL after allogeneic HSCT.