Human plasma cells engineered to secrete bispecifics drive effective in vivo leukemia killing.

Bispecific antibodies are an important tool for the management and treatment of acute leukemias. As a next step toward clinical translation of engineered plasma cells, we describe approaches for secretion of bispecific antibodies by human plasma cells. We show that human plasma cells expressing either fragment crystallizable domain-deficient anti-CD19 × anti-CD3 (blinatumomab) or anti-CD33 × anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of B acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19-bispecific secretion by plasma cells and prevents self-targeting. Plasma cells secreting anti-CD19-bispecific antibodies elicited in vivo control of acute lymphoblastic leukemia patient-derived xenografts in immunodeficient mice co-engrafted with autologous T cells. In these studies, we found that leukemic control elicited by engineered plasma cells was similar to CD19-targeted chimeric antigen receptor-expressing T cells. Finally, the steady-state concentration of anti-CD19 bispecifics in serum 1 month after cell delivery and tumor eradication was comparable with that observed in patients treated with a steady-state infusion of blinatumomab. These findings support further development of ePCs for use as a durable delivery system for the treatment of acute leukemias, and potentially other cancers.

Human plasma cells engineered to secrete bispecifics drive effective in vivo leukemia killing.

Bispecific antibodies are an important tool for the management and treatment of acute leukemias. Advances in genome-engineering have enabled the generation of human plasma cells that secrete therapeutic proteins and are capable of long-term in vivo engraftment in humanized mouse models. As a next step towards clinical translation of engineered plasma cells (ePCs) towards cancer therapy, here we describe approaches for the expression and secretion of bispecific antibodies by human plasma cells. We show that human ePCs expressing either fragment crystallizable domain deficient anti-CD19 × anti-CD3 (blinatumomab) or anti-CD33 × anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of specific primary human cell subsets and B-acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19 bispecific secretion by ePCs and prevents self-targeting. Further, anti-CD19 bispecific-ePCs elicited tumor eradication in vivo following local delivery in flank-implanted Raji lymphoma cells. Finally, immunodeficient mice engrafted with anti-CD19 bispecific-ePCs and autologous T cells potently prevented in vivo growth of CD19+ acute lymphoblastic leukemia in patient-derived xenografts. Collectively, these findings support further development of ePCs for use as a durable, local delivery system for the treatment of acute leukemias, and potentially other cancers.

Activated interleukin-7 receptor signaling drives B-cell acute lymphoblastic leukemia in mice.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype of B-ALL often associated with genetic variants that alter cytokine receptor signaling, including mutations in the interleukin-7 receptor (IL7R). To investigate whether IL7R variants are leukemia-initiating, we built mouse models expressing activated Il7r (aIL7R). B-cell intrinsic aIL7R mice developed spontaneous B-ALL, demonstrating sufficiency of Il7r activating mutations in leukemogenesis. Concomitant introduction of a knock-out allele in the associated adapter protein Lnk (encoded by Sh2b3) or a dominant-negative variant of the transcription factor Ikaros (Ikzf1) increased disease penetrance. The resulting murine leukemias displayed monoclonality and recurrent somatic Kras mutations and efficiently engrafted into immunocompetent mice. Phosphoproteomic analyses of aIL7R leukemic cells revealed constitutive Stat5 signaling and B cell receptor (BCR)-like signaling despite the absence of surface pre-BCR. Finally, in vitro treatment of aIL7R leukemic B-cells with Jak, mTOR, or Syk inhibitors blocked growth, confirming that each pathway is active in this mouse model of IL7R-driven B-ALL.

RNA-binding protein isoforms ZAP-S and ZAP-L have distinct antiviral and immune resolution functions.

The initial response to viral infection is anticipatory, with host antiviral restriction factors and pathogen sensors constantly surveying the cell to rapidly mount an antiviral response through the synthesis and downstream activity of interferons. After pathogen clearance, the host's ability to resolve this antiviral response and return to homeostasis is critical. Here, we found that isoforms of the RNA-binding protein ZAP functioned as both a direct antiviral restriction factor and an interferon-resolution factor. The short isoform of ZAP bound to and mediated the degradation of several host interferon messenger RNAs, and thus acted as a negative feedback regulator of the interferon response. In contrast, the long isoform of ZAP had antiviral functions and did not regulate interferon. The two isoforms contained identical RNA-targeting domains, but differences in their intracellular localization modulated specificity for host versus viral RNA, which resulted in disparate effects on viral replication during the innate immune response.

Activated CARD11 accelerates germinal center kinetics, promoting mTORC1 and terminal differentiation.

Activating mutations in the adapter protein CARD11 associated with diffuse large B cell lymphomas (DLBCLs) are predicted to arise during germinal center (GC) responses, leading to inappropriate activation of NF-κB signaling. Here, we modeled the B cell-intrinsic impact of the L251P activating mutation in CARD11 (aCARD11) on the GC response. Global B cell aCARD11 expression led to a modest increase in splenic B cells and a severe reduction in B1 B cell numbers, respectively. Following T cell-dependent immunization, aCARD11 cells exhibited increased rates of GC formation, resolution, and differentiation. Restriction of aCARD11 to GC B cells similarly altered the GC response and B cell differentiation. In this model, aCARD11 promoted dark zone skewing along with increased cycling, AID levels, and class switch recombination. Furthermore, aCard11 GC B cells displayed increased biomass and mTORC1 signaling, suggesting a novel strategy for targeting aCARD11-driven DLBCL. While aCARD11 potently impacts GC responses, the rapid GC contraction suggests it requires collaboration with events that limit terminal differentiation to promote lymphoma.

CCR7 provides localized access to IL-2 and defines homeostatically distinct regulatory T cell subsets.

Immune tolerance and activation depend on precise control over the number and function of immunosuppressive Foxp3(+) regulatory T (T reg) cells, and the importance of IL-2 in maintaining tolerance and preventing autoimmunity is clear. However, the homeostatic requirement for IL-2 among specific populations of peripheral T reg cells remains poorly understood. We show that IL-2 selectively maintains a population of quiescent CD44(lo)CD62L(hi) T reg cells that gain access to paracrine IL-2 produced in the T cell zones of secondary lymphoid tissues due to their expression of the chemokine receptor CCR7. In contrast, CD44(hi)CD62L(lo)CCR7(lo) T reg cells that populate nonlymphoid tissues do not access IL-2-prevalent regions in vivo and are insensitive to IL-2 blockade; instead, their maintenance depends on continued signaling through the co-stimulatory receptor ICOS (inducible co-stimulator). Thus, we define a fundamental homeostatic subdivision in T reg cell populations based on their localization and provide an integrated framework for understanding how T reg cell abundance and function are controlled by unique signals in different tissue environments.

T-bet(+) Treg cells undergo abortive Th1 cell differentiation due to impaired expression of IL-12 receptor ß2.

Foxp3(+) regulatory T (Treg) cells limit inflammatory responses and maintain immune homeostasis. Although comprised of several phenotypically and functionally distinct subsets, the differentiation of specialized Treg cell populations within the periphery is poorly characterized. We demonstrate that the development of T-bet(+) Treg cells that potently inhibit T helper 1 (Th1) cell responses was dependent on the transcription factor STAT1 and occurred directly in response to interferon-γ produced by effector T cells. Additionally, delayed induction of the IL-12Rß2 receptor component after STAT1 activation helped ensure that Treg cells do not readily complete STAT4-dependent Th1 cell development and lose their ability to suppress effector T cell proliferation. Thus, we define a pathway of abortive Th1 cell development that results in the specialization of peripheral Treg cells and demonstrate that impaired expression of a single cytokine receptor helps maintain Treg cell-suppressive function in the context of inflammatory Th1 cell responses.