Challenges in αCD38-chimeric antigen receptor (CAR)-expressing natural killer (NK) cell-based immunotherapy in multiple myeloma: Harnessing the CD38dim phenotype of cytokine-stimulated NK cells as a strategy to prevent fratricide.

BACKGROUND AIMS: Adoptive cell therapy with chimeric antigen receptor (CAR)-expressing natural killer (NK) cells is an emerging approach that holds promise in multiple myeloma (MM). However, the generation of CAR-NK cells targeting CD38 is met with obstacles due to the expression of CD38 on NK cells. Knock-out of CD38 is currently explored as a strategy, although the consequences of the lack of CD38 expression with regards to engraftment and activity in the bone marrow microenvironment are not fully elucidated. Here, we present an alternative approach by harnessing the CD38dim phenotype occurring during long-term cytokine stimulation of primary NK cells. METHODS: Primary NK cells were expanded from peripheral blood mononuclear cells by long-term IL-2 stimulation. During expansion, the CD38 expression was monitored in order to identify a time point when introduction of a novel affinity-optimized αCD38-CAR confered optimal viability, i.e. prevented fratricide. CD38dim NK cells were trasduced with retroviral vectors encoding for the CAR trasngene and their functionality was assessed in in vitro activation and cytotoxicity assays. RESULTS: We verified the functionality of the αCD38-CAR-NK cells against CD38+ cell lines and primary MM cells. Importantly, we demonstrated that αCD38-CAR-NK cells derived from patients with MM have increased activity against autologous MM samples ex vivo. CONCLUSIONS: Overall, our results highlight that incorporation of a functional αCD38-CAR construct into a suitable NK-cell expansion and activation protocol results in a potent and feasible immunotherapeutic strategy for the treatment of patients with MM.

A Novel Affinity Engineered Anti-CD47 Antibody With Improved Therapeutic Index That Preserves Erythrocytes and Normal Immune Cells.

Therapeutic blockade of the CD47/SIRPα axis by small molecules or monoclonal antibodies (mAbs) is a proven strategy to enhance macrophages-mediated anti-tumor activity. However, this strategy has been hampered by elevated on-target toxicities and rapid clearance due to the extensive CD47 expression on normal cells ("antigen sink") such as red blood cells (RBCs). To address these hurdles, we report on the development of STI-6643, an affinity-engineered fully human anti-CD47 IgG4 antibody with negligible binding to normal cells. STI-6643 exhibited no hemagglutination activity on human RBCs at concentrations up to 300 µg/mL yet specifically blocked the CD47/SIPRα interaction. Of particular interest, STI-6643 preserved T cell functionality in vitro and showed significantly lower immune cell depletion in vivo in contrast to three previously published competitor reference anti-CD47 clones Hu5F9, AO-176 and 13H3. In cynomolgus monkeys, STI-6643 was well-tolerated at the highest dose tested (300 mg/kg/week) and provided favorable clinical safety margins. Finally, STI-6643 displayed comparable anti-tumor activity to the high-affinity reference clone Hu5F9 in a RAJI-Fluc xenograft tumor model as monotherapy or in combination with anti-CD20 (rituximab) or anti-CD38 (daratumumab) mAbs. These data suggest that STI-6643 possesses the characteristics of an effective therapeutic candidate given its potent anti-tumor activity and low toxicity profile.

Therapeutic polyclonal human CD8+ CD25+ Fox3+ TNFR2+ PD-L1+ regulatory cells induced ex-vivo.

We report that polyclonal CD8regs generated in one week ex-vivo with anti-CD3/28 beads and cytokines rapidly developed suppressive activity in vitro sustained by TGF-ß. In immunodeficient mice, these CD8regs demonstrated a markedly protective, IL-10 dependent activity against a xeno-GVHD. They expressed IL-2Rα/ß, Foxp3, TNFR2, and the negative co-stimulatory receptors CTLA-4, PD-1, PD-L1 and Tim-3. Suppressive activity in vitro correlated better with TNFR2 and PD-L1 than Foxp3. Blocking studies suggested that TNF enhanced PD-L1 expression and the suppressive activity of the CD8regs generated. Unlike other polyclonal CD4 and CD8 Tregs, these CD8regs preferentially targeted allogeneic T cells, but they lacked cytotoxic activity against them even after sensitization. Unlike CD4regs, these CD8regs could produce IL-2 and proliferate while inhibiting target cells. If these CD8regs can persist in foreign hosts without impairing immune surveillance, they could serve as a practical remission-inducing product for the treatment of autoimmune diseases, graft-versus-host disease, and allograft rejection.

Natural and TGF-beta-induced Foxp3(+)CD4(+) CD25(+) regulatory T cells are not mirror images of each other.

Foxp3(+) CD4(+) CD25(+) regulatory cell (Treg) subsets that maintain immunologic homeostasis have been considered to be a homogeneous population of naturally occurring, thymus-derived CD4(+)CD25(+) cells (nTregs). However, similar Foxp3+ Tregs can be induced from CD25(-) precursors in vivo, and ex vivo with interleukin 2 (IL-2) and transforming growth factor beta (TGF-beta) (iTregs). These two subsets differ in their principal antigen specificities and in the T-cell receptor signal strength and co-stimulatory requirements needed for their generation. However, whether iTregs have any unique functions in vivo has been unclear. Although IL-6 can convert nTregs to Th17 cells, iTregs induced by IL-2 and TGF-beta are resistant to this cytokine and thereby might retain suppressive function at inflammatory sites. Thus, nTregs and iTregs may have different roles in the adaptive immune response.

Critical role of IL-2 and TGF-beta in generation, function and stabilization of Foxp3+CD4+ Treg.

CD4+Foxp3+ Treg consist of two indistinguishable subsets induced in either the thymus or the periphery. In addition to their suppressive activities, IL-6 can convert natural Treg to pro-inflammatory IL-17-producing cells, but those induced with IL-2 and TGF-beta remain Treg. Unlike mouse CD4+CD25(-) cells, which rapidly become polyclonal Foxp3+CD25+ Treg when activated appropriately with IL-2 and TGF-beta, human T cells require multiple stimulations to become similar suppressor cells.

IL-2 is essential for TGF-beta to convert naive CD4+CD25- cells to CD25+Foxp3+ regulatory T cells and for expansion of these cells.

IL-2 and TGF-beta both have important roles in the induction and maintenance of immunologic tolerance, but whether these cytokines act separately or together to achieve this effect is poorly understood. Although others have reported that IL-2 can directly enhance forkhead box protein P3 (Foxp3) transcription factor expression by natural CD4(+)CD25(+) regulatory T cells, in this study, we report that the role of IL-2 on the generation of peripheral regulatory CD4(+) cells is indirect. Ab neutralization studies and experiments with IL-2-deficient mice have revealed that IL-2 is required for TGF-beta to induce naive CD4(+)CD25(-) cells to become CD25(+) and express Foxp3, and develop the characteristic properties of CD4(+)CD25(+) regulatory cells. This effect of IL-2 on the generation and expansion of these adaptive Foxp3(+) regulatory cells is nonredundant, but IL-4, IL-7, and IL-15, other common gamma-chain cytokines, could sustain Foxp3 expression. Because subjects with autoimmune diseases often have defects in the production of IL-2 and/or TGF-beta, the generation of autologous T regulatory cells ex vivo with these cytokines for transfer in vivo may have considerable therapeutic potential.

TGF-beta requires CTLA-4 early after T cell activation to induce FoxP3 and generate adaptive CD4+CD25+ regulatory cells.

Although positive CD28 costimulation is needed for the generation of natural CD4+CD25+ regulatory T cells, we report that negative CTLA-4 costimulation is necessary for generating phenotypically and functionally similar adaptive CD4+CD25+ suppressor cells. TGF-beta could not induce CD4+CD25- cells from CTLA-4(-/-) mice to express normal levels of FoxP3 or to develop suppressor activity. Moreover, blockade of CTLA-4 following activation of wild-type CD4+ cells abolished the ability of TGF-beta to induce FoxP3-expressing mouse suppressor cells. TGF-beta accelerated expression of CTLA-4, and time course studies suggested that CTLA-4 ligation of CD80 shortly after T cell activation enables TGF-beta to induce CD4+CD25- cells to express FoxP3 and develop suppressor activity. TGF-beta also enhanced CD4+ cell expression of CD80. Thus, CTLA-4 has an essential role in the generation of acquired CD4+CD25+ suppressor cells in addition to its other inhibitory effects. Although natural CD4+CD25+ cells develop normally in CTLA-4(-/-) mice, the lack of TGF-beta-induced, peripheral CD4+CD25+ suppressor cells in these mice may contribute to their rapid demise.

Transfer of regulatory T cells generated ex vivo modifies graft rejection through induction of tolerogenic CD4+CD25+ cells in the recipient.

Certain CD4+CD25+ T cells can induce and maintain T-cell non-responsiveness to donor alloantigens and have therapeutic potential in solid organ transplantation. Peripheral CD4+CD25- cells alloactivated with IL-2 and transforming growth factor beta (TGF-beta) ex vivo express the transcription factor FoxP3, and become potent antigen-specific CD4+CD25- suppressor cells. Here we report that the transfer of TGF-beta-induced regulatory CD4+ and CD8+ T cells (Tregs) co-incident with transplantation of a histoincompatible heart resulted in extended allograft survival. To account for this result, we injected non-transplanted mice with a single dose of CD4+ and CD8+ Tregs and transferred donor cells every 2 weeks to mimic the continuous stimulation of a transplant. We observed increased splenic CD4+CD25+ cells that were of recipient origin. These cells rendered the animals non-responsive to donor alloantigens by an antigen-specific and cytokine-dependent mechanism of action. Both the increased number of CD4+CD25+ cells and their tolerogenic effect were dependent on continued donor antigen boosting. Thus, Tregs generated ex vivo can act like a vaccine that generates host suppressor cells with the potential to protect MHC-mismatched organ grafts from rejection.

Natural and induced CD4+CD25+ cells educate CD4+CD25- cells to develop suppressive activity: the role of IL-2, TGF-beta, and IL-10.

Thymus-derived, natural CD4(+)CD25(+) regulatory T cells can educate peripheral CD4(+)CD25(-) cells to develop suppressive activity by poorly understood mechanisms. TGF-beta has IL-2-dependent costimulatory effects on alloactivated naive, human CD4(+) T cells and induces them ex vivo to become potent contact-dependent, cytokine-independent suppressor cells. In this study, we report that CD4(+)CD25(+) cells are the targets of the costimulatory effects of IL-2 and TGF-beta. These cells do not divide, but, instead, greatly increase the numbers of CD4(+)CD25(-) cells that become CD25(+) cytokine-independent suppressor cells. These CD4(+)CD25(+) regulatory cells, in turn, induce other alloactivated CD4(+)CD25(-) cells to become potent suppressor cells by mechanisms that, surprisingly, require both cell contact and TGF-beta and IL-10. The suppressive effects of these secondary CD4(+)CD25(+) cells depend upon TGF-beta and IL-10. Moreover, both the naive CD4(+) cells induced by IL-2 and TGF-beta to become suppressor cells, and the subsequent CD4(+)CD25(-) cells educated by them to become suppressors express FoxP3. We suggest that the long-term effects of adoptively transferred natural-like CD4(+)CD25(+) regulatory cells induced ex vivo are due to their ability to generate new cytokine-producing CD4(+) regulatory T cells in vivo.

Regulatory T cells generated ex vivo as an approach for the therapy of autoimmune disease.

Regulatory T cells control the reactivity of potentially harmful, self-reactive T cells and prevent autoimmune diseases. Significant progress has been made in the identification, derivation, and mechanism of action of T regulatory cells, previously called suppressor T cells. Heterogeneous T regulatory subsets can be grouped into naturally occurring and those induced in the periphery. Here, we consider whether we can harness T regulatory cells to function as a therapeutic agent for patients with established autoimmune diseases. Since the principal function of thymus-derived, natural CD4+CD25+ cells is to prevent autoimmunity, this subset would be an obvious choice. Besides their contact-dependent, cytokine-independent mechanism of action, they can also induce other CD4+ cells to become suppressor cells. However, only few natural CD4+CD25+ cells circulate in human peripheral blood. Alternatively, one can use IL-2 and TGF-beta to generate large numbers of CD4+CD25+ regulatory T cells ex vivo from naive T cells. These cells have the phenotypic and functional properties similar to natural CD4+CD25+ cells, including the capacity to induce CD4+CD25- cells to develop suppressive activity. These natural-like CD4+CD25+ regulatory T cells are the product of separate effects of IL-2 and TGF-beta on both natural CD4+CD25+ and CD4+CD25- cells. The ability of natural-like CD4+CD25+ cells to induce other CD4+CD25- cells to develop suppressive activity is both contact-dependent and cytokine-dependent. Thus, the effects of IL-2 and TGF-beta on both natural CD4+CD25+ cells and CD4+CD25- cells may trigger a continuous loop which results in the renewal of antigen-specific CD4+ regulatory T cells. These studies suggest that the adoptive transfer of CD4+ T regulatory cells generated ex vivo with IL-2 and TGF-beta as a treatment for autoimmune diseases may have sustained, long-term beneficial effects.

CD4+ and CD8+ regulatory T cells generated ex vivo with IL-2 and TGF-beta suppress a stimulatory graft-versus-host disease with a lupus-like syndrome.

Regulatory T cells generated ex vivo from conventional mouse T cells have been used to prevent and alter the course of a stimulatory graft-vs-host disease with a lupus-like syndrome. DBA/2 mouse T cells induce this syndrome when injected into (DBA/2 x C57BL/6) F(1) mice. Stimulating DBA/2 T cells with irradiated C57BL/6 in the presence of IL-2 and TGF-beta induced both CD4(+) and CD8(+) cells to develop potent suppressive activity and enhanced their survival. The IL-2 and TGF-beta-treated T cells lost their ability to induce graft-vs-host disease and, instead, prevented other parental T cells from inducing lymphoid hyperplasia, B cell activation, and an immune complex glomerulonephritis. Moreover, a single transfer of TGF-beta-conditioned T cells to animals that had already developed anti-dsDNA Abs decreased the titer, suppressed proteinuria, and doubled survival. This study raises the possibility that autologous regulatory T cells generated ex vivo have the potential to be used as an adoptive immunotherapy to induce allograft tolerance and to control autoimmunity.

The role of the combination of IL-2 and TGF-beta or IL-10 in the generation and function of CD4+ CD25+ and CD8+ regulatory T cell subsets.

Recently, considerable attention has been focused on thymus-derived CD4+ regulatory T cells that constitutively express CD25 and have a contact-dependent, cytokine-independent mechanism in vitro. However, peripheral CD4+ and CD8+ T cells can also be induced to become regulatory T cells. Here we review our studies using the combination of IL-2 and transforming growth factor beta (TGF-beta) to generate regulatory T cell subsets ex vivo, and the work of others using IL-10 to induce suppressive activity. Under certain conditions, the autocrine effects of TGF-beta and IL-10 induce peripheral T cells to produce immunosuppressive levels of each of these cytokines. This effect of TGF-beta is IL-2 dependent. Under other conditions IL-2 and TGF-beta can induce CD4+ cells to develop potent contact-dependent, cytokine-independent regulatory activity. At present, there is considerable confusion concerning the mechanism of action of CD4+ CD25+ cells because cytokine-producing regulatory T cells generated in the periphery can express CD25 and other markers displayed by naturally occurring, thymus-derived regulatory T cells. We, therefore, propose a nomenclature that identifies thymus-derived and peripheral regulatory cells, and that also differentiates T regulatory cells from T helper cells. Because T regulatory cells broadly control T helper cell reactivity, the mechanisms that control regulatory cell function are also reviewed. Finally, the potential use of regulatory T cells generated ex vivo as an adoptive immunotherapy for certain autoimmune diseases, to prevent organ graft rejection, or to prevent pathologic host responses to infectious agents is discussed.

Generation ex vivo of TGF-beta-producing regulatory T cells from CD4+CD25- precursors.

Previously we reported that TGF-beta has an important role in the generation and expansion of human "professional" CD4(+)CD25(+) regulatory T cells in the periphery that have a cytokine-independent mechanism of action. In this study we used low-dose staphylococcal enterotoxin to induce T cell-dependent Ab production. We report that TGF-beta induces activated CD4(+)CD25(-) T cells to become Th3 suppressor cells. While stimulating CD4(+) cells with TGF-beta modestly increased expression of CD25 and intracellular CTLA-4 in primary cultures, upon secondary stimulation without TGF-beta the total number and those expressing these markers dramatically increased. This expansion was due to both increased proliferation and protection of these cells from activation-induced apoptosis. Moreover, adding as few as 1% of these TGF-beta-primed CD4(+) T cells to fresh CD4(+) cells and B cells markedly suppressed IgG production. The inhibitory effect was mediated by TGF-beta and was also partially contact dependent. Increased TGF-beta production was associated with a decreased production of IFN-gamma and IL-10. Depletion studies revealed that the precursors of these TGF-beta-producing CD4(+) suppressor cells were CD25 negative. These studies provide evidence that CD4(+)CD25(+) regulatory cells in human blood consist of at least two subsets that have TGF-beta-dependent and independent mechanisms of action. TGF-beta has an essential role in the generation of both of these T suppressor cell subsets from peripheral T cells. The ability to induce CD4(+) and CD8(+) cells to become regulatory cells ex vivo has the potential to be useful in the treatment of autoimmune diseases and to prevent transplant rejection.

The potential of human regulatory T cells generated ex vivo as a treatment for lupus and other chronic inflammatory diseases.

Regulatory T cells prevent autoimmunity by suppressing the reactivity of potentially aggressive self-reactive T cells. Contact-dependent CD4+ CD25+ 'professional' suppressor cells and other cytokine-producing CD4+ and CD8+ T-cell subsets mediate this protective function. Evidence will be reviewed that T cells primed with transforming growth factor (TGF)-beta expand rapidly following restimulation. Certain CD4+ T cells become contact-dependent suppressor cells and other CD4+ and CD8+ cells become cytokine-producing regulatory cells. This effect is dependent upon a sufficient amount of IL-2 in the microenvironment to overcome the suppressive effects of TGF-beta. The adoptive transfer of these suppressor cells generated ex vivo can protect mice from developing chronic graft-versus-host disease with a lupus-like syndrome and alter the course of established disease. These data suggest that autologous T cells primed and expanded with TGF-beta have the potential to be used as a therapy for patients with systemic lupus erythematosus and other chronic inflammatory diseases. This novel adoptive immunotherapy also has the potential to prevent the rejection of allogeneic transplants.