Immune checkpoint blockade therapy has been successful in treating some types of cancers but has not shown clinical benefits for treating leukemia 1 . This result suggests that leukemia exploits unique escape mechanisms. Certain immune inhibitory receptors that are expressed by normal immune cells are also present on leukemia cells. It remains unknown whether these receptors can initiate immune-related primary signaling in tumor cells. Here we show that LILRB4, an ITIM-containing receptor and a monocytic leukemia marker, supports tumor cell infiltration into tissues and suppresses T cell activity via ApoE/LILRB4/SHP-2/uPAR/Arginase-1 signaling axis in acute myeloid leukemia (AML) cells. Blocking LILRB4 signaling using knockout and antagonistic antibody approaches impeded AML development. Thus, LILRB4 orchestrates tumor invasion pathways in monocytic leukemia cells by creating an immune-suppressive microenvironment. LILRB4 represents a compelling target for treatment of monocytic AML.

Inhibitory leukocyte immunoglobulin-like receptors (LILRBs 1-5) transduce signals via intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that recruit protein tyrosine phosphatase non-receptor type 6 (PTPN6 or SHP-1), protein tyrosine phosphatase non-receptor type 11 (PTPN11 or SHP-2), or Src homology 2 domain-containing inositol phosphatase (SHIP), leading to negative regulation of immune cell activation. Certain of these receptors also play regulatory roles in neuronal activity and osteoclast development. The activation of LILRBs on immune cells by their ligands may contribute to immune evasion by tumors. Recent studies found that several members of LILRB family are expressed by tumor cells, notably hematopoietic cancer cells, and may directly regulate cancer development and relapse as well as the activity of cancer stem cells. LILRBs thus have dual concordant roles in tumor biology – as immune checkpoint molecules and as tumor-sustaining factors. Importantly, the study of knockout mice indicated that LILRBs do not affect hematopoiesis and normal development. Therefore LILRBs may represent ideal targets for tumor treatment. This review aims to summarize current knowledge on expression patterns, ligands, signaling, and functions of LILRB family members in the context of cancer development.

PURPOSE CD19-targeted chimeric antigen receptor T cells (CD19-CAR) and blinatumomab effectively induce remission in relapsed or refractory B-cell acute lymphoblastic leukemia (ALL) but are also associated with CD19 antigen modulation. There are limited data regarding the impact of prior blinatumomab exposure on subsequent CD19-CAR outcomes. PATIENTS AND METHODS We conducted a multicenter, retrospective review of children and young adults with relapsed or refractory ALL who received CD19-CAR between 2012 and 2019. Primary objectives addressed 6-month relapse-free survival (RFS) and event-free survival (EFS), stratified by blinatumomab use. Secondary objectives included comparison of longer-term survival outcomes, complete remission rates, CD19 modulation, and identification of factors associated with EFS. RESULTS Of 420 patients (median age, 12.7 years; interquartile range, 7.1-17.5) treated with commercial tisagenlecleucel or one of three investigational CD19-CAR constructs, 77 (18.3%) received prior blinatumomab. Blinatumomab-exposed patients more frequently harbored KMT2A rearrangements and underwent a prior stem-cell transplant than blinatumomab-naïve patients. Among patients evaluable for CD19-CAR response (n = 412), blinatumomab nonresponders had lower complete remission rates to CD19-CAR (20 of 31, 64.5%) than blinatumomab responders (39 of 42, 92.9%) or blinatumomab-naive patients (317 of 339, 93.5%), P < .0001. Following CD19-CAR, blinatumomab nonresponders had worse 6-month EFS (27.3%; 95% CI, 13.6 to 43.0) compared with blinatumomab responders (66.9%; 95% CI, 50.6 to 78.9; P < .0001) or blinatumomab-naïve patients (72.6%; 95% CI, 67.5 to 77; P < .0001) and worse RFS. High-disease burden independently associated with inferior EFS. CD19-dim or partial expression (preinfusion) was more frequently seen in blinatumomab-exposed patients (13.3% v 6.5%; P = .06) and associated with lower EFS and RFS. CONCLUSION With the largest series to date in pediatric CD19-CAR, and, to our knowledge, the first to study the impact of sequential CD19 targeting, we demonstrate that blinatumomab nonresponse and high-disease burden were independently associated with worse RFS and EFS, identifying important indicators of long-term outcomes following CD19-CAR.

To effectively improve treatment for acute myeloid leukemia (AML), new molecular targets and therapeutic approaches need to be identified. Chimeric antigen receptor (CAR)-modified T cells targeting tumor-associated antigens have shown promise in the treatment of some malignancies. However, CAR-T cell development for AML has been limited by lack of an antigen with high specificity for AML cells that is not present on normal hematopoietic stem cells, and thus will not result in myelotoxicity. Here we demonstrate that leukocyte immunoglobulin-like receptor-B4 (LILRB4) is a tumor-associated antigen highly expressed on monocytic AML cells. We generated a novel anti-LILRB4 CAR-T cell that displays high antigen affinity and specificity. These CAR-T cells display efficient effector function in vitro and in vivo against LILRB4 AML cells. Furthermore, we demonstrate anti-LILRB4 CAR-T cells are not toxic to normal CD34 umbilical cord blood cells in colony-forming unit assays, nor in a humanized hematopoietic-reconstituted mouse model. Our data demonstrate that anti-LILRB4 CAR-T cells specifically target monocytic AML cells with no toxicity to normal hematopoietic progenitors. This work thus offers a new treatment strategy to improve outcomes for monocytic AML, with the potential for elimination of leukemic disease while minimizing the risk for on-target off-tumor toxicity.

Relapse following chimeric antigen receptor (CAR) T-cell therapy directed against CD19 for relapsed/refractory B-acute lymphoblastic leukemia (r/r B-ALL) remains a significant challenge. Three main patterns of relapse predominate: CD19 positive (CD19pos) relapse, CD19 negative (CD19neg) relapse, and lineage switch (LS). Development and validation of risk factors that predict relapse phenotype could help define potential pre- or post-CAR T-cell infusion interventions aimed at decreasing relapse. Our group sought to extensively characterize pre-infusion risk factors associated with the development of each relapse pattern via a multicenter, retrospective review of children and young adults with r/r B-ALL treated with a murine-based CD19-CAR construct. Of 420 CAR-treated patients, 166 (39.5%) relapsed, including 83 (50%) CD19pos, 68 (41%) CD19neg, and 12 (7.2%) LS relapses. A greater cumulative number of prior complete remissions was associated with CD19pos relapses, whereas high pre-infusion disease burden, prior blinatumomab non-response, older age, and 4-1BB CAR construct were associated with CD19neg relapses. The presence of a KMT2A rearrangement was the only pre-infusion risk factor associated with LS. The median overall survival following a post-CAR relapse was 11.9 months (95% CI 9-17 months) and was particularly dismal in patients experiencing a LS, with no long-term survivors following this pattern of relapse. Given the poor outcomes for those with post-CAR relapse, study of relapse prevention strategies, such as consolidative hematopoietic stem cell transplant, is critical and warrants further investigation on prospective clinical trials.

BackgroundCurrent immune checkpoint blockade strategies have been successful in treating certain types of solid cancer. However, checkpoint blockade monotherapies have not been successful against most hematological malignancies including multiple myeloma and leukemia. There is an urgent need to identify new targets for development of cancer immunotherapy. LILRB1, an immunoreceptor tyrosine-based inhibitory motif-containing receptor, is widely expressed on human immune cells, including B cells, monocytes and macrophages, dendritic cells and subsets of natural killer (NK) cells and T cells. The ligands of LILRB1, such as major histocompatibility complex (MHC) class I molecules, activate LILRB1 and transduce a suppressive signal, which inhibits the immune responses. However, it is not clear whether LILRB1 blockade can be effectively used for cancer treatment.MethodsFirst, we measured the LILRB1 expression on NK cells from cancer patients to determine whether LILRB1 upregulated on NK cells from patients with cancer, compared with NK cells from healthy donors. Then, we developed specific antagonistic anti-LILRB1 monoclonal antibodies and studied the effects of LILRB1 blockade on the antitumor immune function of NK cells, especially in multiple myeloma models, in vitro and in vivo xenograft model using non-obese diabetic (NOD)-SCID interleukin-2Rγ-null mice.ResultsWe demonstrate that percentage of LILRB1+ NK cells is significantly higher in patients with persistent multiple myeloma after treatment than that in healthy donors. Further, the percentage of LILRB1+ NK cells is also significantly higher in patients with late-stage prostate cancer than that in healthy donors. Significantly, we showed that LILRB1 blockade by our antagonistic LILRB1 antibody increased the tumoricidal activity of NK cells against several types of cancer cells, including multiple myeloma, leukemia, lymphoma and solid tumors, in vitro and in vivo.ConclusionsOur results indicate that blocking LILRB1 signaling on immune effector cells such as NK cells may represent a novel strategy for the development of anticancer immunotherapy.

Leukocyte immunoglobulin-like receptor B (LILRB), a family of immune checkpoint receptors, contribute to acute myeloid leukemia (AML) development, but the specific mechanisms triggered by activation or inhibition of these immune checkpoints in cancer is largely unknown. Here we demonstrated that the intracellular domain of LILRB3 is constitutively associated with the adaptor protein TRAF2. Activated LILRB3 in AML cells leads to recruitment of cFLIP and subsequent NF-κB upregulation, resulting in enhanced leukemic cell survival and inhibition of T cell-mediated anti-tumor activity. Hyperactivation of NF-κB induces a negative regulatory feedback loop mediated by A20, which disrupts the interaction of LILRB3 and TRAF2; consequently the SHP-1/2-mediated inhibitory activity of LILRB3 becomes dominant. Finally, we show that blockade of LILRB3 signaling with antagonizing antibodies hampers AML progression. LILRB3 thus exerts context-dependent activating and inhibitory functions, and targeting LILRB3 may become a potential therapeutic strategy for AML treatment.

Introduction: Chimeric antigen receptor (CAR) T-cells redirected against CD19 have demonstrated remarkable clinical activity in children and adults with relapsed/refractory (r/r) B-cell malignancies. The risk of lineage switch (LS) following CD19-directed therapies has been well documented but has been primarily limited to case reports. Additionally, the risk of subsequent malignant neoplasms (SMN) following CAR T-cells has not yet been described. Distinguishing LS (B-ALL to myeloid malignancy) from a therapy-related myeloid neoplasm is both clinically and biologically relevant. The former emerges from a highly refractory leukemic clone, likely resistant to salvage therapy, whereas the latter represents a new malignancy that can be associated with long-term survival. Methods: We conducted a multicenter, retrospective review of children and young adults with r/r B-acute lymphoblastic leukemia (B-ALL) who received either commercial tisagenlecleucel or 1 of 3 investigational murine-based CD19-CAR constructs on clinical trials at 7 US centers between 2012-2019. Patients diagnosed with B-ALL before age 25 years were included and patients who had received any prior CAR product were excluded. Results: Of 420 CAR-treated patients, with a median follow-up of 30.1 months, 12 (2.9%) experienced LS and 6 (1.4%) developed a SMN (Table). The median time to diagnosis of LS following CAR T-cell infusion was significantly shorter compared to diagnosis of SMN (65.5 days vs. 883.5 days; p=0.005). Eleven of 12 patients (91.7%) with LS converted to acute myeloid leukemia (AML). One patient converted to mixed phenotype acute leukemia, B/myeloid type. The leukemia of 10 of 12 patients with LS harbored cytogenetics similar to those at initial diagnosis. For the remaining 2 patients with LS, cytogenetics were unavailable, but the leukemias were considered LS by the treating institution. KMT2Ar rearrangement (KMT2Ar) was a predominant cytogenetic abnormality seen in patients with LS. Overall, 38 of 420 patients (9%) had a KMT2Ar. KMT2Ar was present in 9 of 12 (75%) patients with LS compared to 20 of 408 (7.1%) non-LS patients (p&lt;0.001). Patients with LS were younger at initial diagnosis compared to the remaining cohort (median age, 1.6 years vs. 7.7 years; p=0.001), reflecting the inherent association between KMT2Ar and infant ALL. Otherwise, there were no significant differences in gender, prior hematopoietic stem cell transplant (HSCT), prior blinatumomab exposure, or CAR response. Within the KMT2Ar cohort, 31 (81.6%) patients achieved a complete remission post-CAR. Eight of these patients received a consolidative HSCT (representing 4 first and 4 second HSCTs). No KMT2Ar patient experienced a post-HSCT LS and 3 are alive with a median follow-up of 1164 days post-CAR. In contrast, of the 23 KTM2Ar patients who did not receive HSCT post-CAR, 7 developed LS and 14 are alive with a median follow-up of 864 days post-CAR. Relative contraindications to post-CAR HSCT included a prior HSCT (n=11) or early LS (n=5). Of the 7 CAR non-responding patients with KMT2Ar, 2 (28.6%) had rapid emergence of LS by the first restaging timepoint. There are no long-term survivors following LS, regardless of KMT2A status, dying a median of 123 days (range, 36-594 days) after diagnosis of LS. The 6 SMNs were cholangiocarcinoma, synovial sarcoma, malignant melanoma and 3 therapy-related myeloid neoplasms (MDS/AML), distinguished from LS based on loss of original cytogenetics. Notably, 4/6 (67%) patients that developed a SMN had received an allogeneic HSCT prior to development of SMN. Four patients (67%) remain alive and in remission with a median follow-up of 304 days after diagnosis of SMN, including 2 patients with MDS/AML. Conclusions: In the largest series of pediatric patients treated with CAR T-cell therapy, we show that LS occurs in 2.9% of children. The presence of a KMT2Ar was the biggest risk factor, with 23.7% of these patients experiencing LS. We found that LS can occur very early in a patient's post CAR T-cell course, and despite a variety of treatment approaches, the outcomes for these patients are dismal. Given the predisposition to LS, the role for consolidative HSCT in KMT2Ar patients warrants further study. Limited by a short follow-up period, we saw SMNs in only 1.4% of our patients. Causality is unknown and likely unrelated to CAR-T, but this further supports the long-term safety of CAR T-cells in children with B-ALL. Figure 1 Figure 1. Disclosures Borowitz: Amgen, Blueprint Medicines: Honoraria. Lee: Harpoon Therapeutics: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Other: trial funding; Gilead: Other: trial funding. Grupp: Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Kite, Vertex, and Servier: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Verneris: jazz: Other: advisory board; Novartis: Other: advisory board; Fate Therapeutics: Consultancy. Gore: Mirati: Current equity holder in publicly-traded company; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Honoraria; Clovis: Current equity holder in publicly-traded company; Celgene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Sanofi Paris: Current equity holder in publicly-traded company; Anchiano: Current equity holder in publicly-traded company; Blueprint Medicines: Current equity holder in publicly-traded company. Brown: Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Kura: Membership on an entity's Board of Directors or advisory committees; KIte: Membership on an entity's Board of Directors or advisory committees. Pulsipher: Equillium: Membership on an entity's Board of Directors or advisory committees; Adaptive: Research Funding; Jasper Therapeutics: Honoraria. Rheingold: Pfizer: Research Funding; Optinose: Other: Spouse's current employment. Gardner: BMS: Patents & Royalties; Novartis: Consultancy.