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The clinical benefit of CTLA-4 blockade on T cells is known, yet the impact of its expression on cancer cells remains unaddressed. We define an immunosuppressive role for tumor-expressed CTLA-4 using chronic lymphocytic leukemia (CLL) as a disease model. CLL cells, among other cancer cells, are CTLA-4+. Coculture with activated human T cells induced surface CTLA-4 on primary human CLL B cells. CTLA-4 on CLL-derived human cell lines decreased CD80 expression on cocultured CD80+ cells, with restoration upon CTLA-4 blockade. Coculture of CTLA-4+ CLL cells with CD80-GFP+ cell lines revealed transfer of CD80-GFP into CLL tumor cells, similar to CTLA-4+ T cells able to trans-endocytose CD80. Coculture of T cells with CTLA-4+ CLL cells decreased IL-2 production. Using a human CTLA-4 knock-in mouse lacking FcγR function, antitumor efficacy was observed by blocking murine CTLA-4 on tumor cells in isolation of the T cell effect and Fc-mediated depletion. These data implicate tumor CTLA-4 in cancer cell–mediated immunosuppression in vitro and as having a functional role in tumor cells in vivo.

Antibody-drug conjugates directed against tumor-specific targets have allowed targeted delivery of highly potent chemotherapy to malignant cells while sparing normal cells. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein with limited expression on normal adult tissues and is overexpressed on the surface of malignant cells in mantle cell lymphoma, acute lymphocytic leukemia with t(1;19)(q23;p13) translocation, and chronic lymphocytic leukemia. This differential expression makes ROR1 an attractive target for antibody-drug conjugate therapy, especially in malignancies such as mantle cell lymphoma and acute lymphocytic leukemia, in which systemic chemotherapy remains the gold standard. Several preclinical and phase 1 clinical studies have established the safety and effectiveness of anti-ROR1 monoclonal antibody–based therapies. Herein we describe a humanized, first-in-class anti-ROR1 antibody-drug conjugate, huXBR1-402-G5-PNU, which links a novel anti-ROR1 antibody (huXBR1-402) to a highly potent anthracycline derivative (PNU). We found that huXBR1-402-G5-PNU is cytotoxic to proliferating ROR1+ malignant cells in vitro and suppressed leukemia proliferation and extended survival in multiple models of mice engrafted with human ROR1+ leukemia. Lastly, we show that the B-cell lymphoma 2 (BCL2)-dependent cytotoxicity of huXBR1-402-G5-PNU can be leveraged by combined treatment strategies with the BCL2 inhibitor venetoclax. Together, our data present compelling preclinical evidence for the efficacy of huXBR1-402-G5-PNU in treating ROR1+ hematologic malignancies.

Acute myeloid leukemia (AML) is the second most common type of leukemia in adults. Incidence of AML increases with age with a peak incidence at 67 years. Patients older than 60 years have an unfavorable prognosis due to resistance to conventional chemotherapy. Volasertib (BI 6727) is a cell-cycle regulator targeting polo-like kinase which has been evaluated in clinical trials in AML. We evaluated effects of volasertib in primary patient samples and NK cells. At equivalent doses, volasertib is cytotoxic to AML blasts but largely spares healthy NK cells. We then evaluated the effect of volasertib treatment in combination with BI 836858 on primary AML blast samples using antibody-dependent cellular cytotoxicity (ADCC) assays. Volasertib treatment of NK cells did not impair NK function as evidenced by comparable levels of BI 836858 mediated ADCC in both volasertib-treated and control-treated NK cells. In summary, volasertib is cytotoxic to AML blasts while sparing NK cell viability and function. Higher BI 836858 mediated ADCC was observed in patient samples pretreated with volasertib. These findings provide a strong rationale to test combination of BI 836858 and volasertib in AML.

The clinical success of ibrutinib validates Bruton tyrosine kinase (BTK) inhibition as an effective strategy for treating hematologic malignancies, including chronic lymphocytic leukemia (CLL). Despite ibrutinib’s ability to produce durable remissions in patients, acquired resistance can develop, mostly commonly by mutation of C481 of BTK in the ibrutinib binding site. Here, we characterize a novel BTK inhibitor, GDC-0853, to evaluate its preclinical efficacy in ibrutinib-naive and ibrutinib-resistant CLL. GDC-0853 is unique among reported BTK inhibitors in that it does not rely upon covalent reaction with C481 to stabilize its occupancy within BTK’s adenosine triphosphate binding site. As with ibrutinib, GDC-0853 potently reduces B-cell receptor signaling, viability, NF-κB–dependent transcription, activation, and migration in treatment naive CLL cells. We found that GDC-0853 also inhibits the most commonly reported ibrutinib-resistant BTK mutant (C481S) both in a biochemical enzyme activity assay and in a stably transfected 293T cell line and maintains cytotoxicity against patient CLL cells harboring C481S BTK mutations. Additionally, GDC-0853 does not inhibit endothelial growth factor receptor or ITK, 2 alternative targets of ibrutinib that are likely responsible for some adverse events and may reduce the efficacy of ibrutinib-antibody combinations, respectively. Our results using GDC-0853 indicate that noncovalent, selective BTK inhibition may be effective in CLL either as monotherapy or in combination with therapeutic antibodies, especially among the emerging population of patients with acquired resistance to ibrutinib therapy.

The Bruton tyrosine kinase inhibitor (BTKi) ibrutinib has transformed chronic lymphocytic leukemia (CLL) therapy but requires continuous administration. These factors have spurred interest in combination treatments. Unlike with chemotherapy, CD20-directed antibody therapy has not improved the outcome of BTKi treatment. Whereas CD20 antigen density on CLL cells decreases during ibrutinib treatment, the B-cell activating factor (BAFF) and its receptor (BAFF-R) remain elevated. Furthermore, BAFF signaling via noncanonical NF-κB remains elevated with BTKi treatment. Blocking BAFF interaction with BAFF-R by using VAY-736, a humanized defucosylated engineered antibody directed against BAFF-R, antagonized BAFF-mediated apoptosis protection and signaling at the population and single-cell levels in CLL cells. Furthermore, VAY-736 showed superior antibody-dependent cellular cytotoxicity compared with CD20- and CD52-directed antibodies used in CLL. VAY-736 exhibited in vivo activity as a monotherapy and, when combined with ibrutinib, produced prolonged survival compared with either therapy alone. The in vivo activity of VAY-736 is dependent upon immunoreceptor tyrosine–based activation motif (ITAM)–mediated activation of effector cells as shown by using an ITAM-deficient mouse model. Collectively, our findings support targeting the BAFF signaling pathway with VAY-736 to more effectively treat CLL as a single agent and in combination with ibrutinib.

Fcγ receptors (FcγR) are critical mediators of monoclonal antibody cancer therapies, as they drive cytotoxic processes upon binding of effector cells to opsonized targets. Along with natural killer (NK) cells, monocytes are also known to destroy antibody-coated targets via antibody-dependent cellular cytotoxicity (ADCC). However, the precise mechanisms by which monocytes carry out this function have remained elusive. Here, we show that human monocytes produce the protease Granzyme B upon both FcγR and Toll-like Receptor (TLR) 8 activation. Treatment with TLR8 agonists elicited Granzyme B and also enhanced FcγR-mediated Granzyme B production in an additive fashion. Furthermore, monocyte-mediated ADCC against cetuximab-coated tumor targets was enhanced by TLR8 agonist treatment, and this enhancement of ADCC required Granzyme B. Hence, we have identified Granzyme B as an important mediator of FcγR function in human monocytes and have uncovered another mechanism by which TLR8 agonists may enhance FcγR-based therapies.

BACKGROUND. Ibrutinib has been shown to have immunomodulatory effects by inhibiting Bruton’s tyrosine kinase (BTK) and IL-2–inducible T cell kinase (ITK). The relative importance of inhibiting these 2 kinases has not been examined despite its relevance to immune-based therapies. METHODS. Peripheral blood mononuclear cells from chronic lymphocytic leukemia (CLL) patients on clinical trials of ibrutinib (BTK/ITK inhibitor; n = 19) or acalabrutinib (selective BTK inhibitor; n = 13) were collected serially. T cell phenotype, immune function, and CLL cell immunosuppressive capacity were evaluated. RESULTS. Ibrutinib markedly increased CD4+ and CD8+ T cell numbers in CLL patients. This effect was more prominent in effector/effector memory subsets and was not observed with acalabrutinib. Ex vivo studies demonstrated that this may be due to diminished activation-induced cell death through ITK inhibition. PD-1 and CTLA-4 expression was significantly markedly reduced in T cells by both agents. While the number of Treg cells remained unchanged, the ratio of these to conventional CD4+ T cells was reduced with ibrutinib, but not acalabrutinib. Both agents reduced expression of the immunosuppressive molecules CD200 and BTLA as well as IL-10 production by CLL cells. CONCLUSIONS. Ibrutinib treatment increased the in vivo persistence of activated T cells, decreased the Treg/CD4+ T cell ratio, and diminished the immune-suppressive properties of CLL cells through BTK-dependent and -independent mechanisms. These features provide a strong rationale for combination immunotherapy approaches with ibrutinib in CLL and other cancers. TRIAL REGISTRATION. ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01589302","term_id":"NCT01589302"}}NCT01589302 and {"type":"clinical-trial","attrs":{"text":"NCT02029443","term_id":"NCT02029443"}}NCT02029443. Samples described here were collected per OSU-0025. FUNDING. The National Cancer Institute.

Breast cancer is the second leading cause of cancer-related deaths in women. The need for new clinical biomarkers in breast cancer is necessary to further predict prognosis and therapeutic response. In this article, the LC-MS histone H1 phosphorylation profiles were established for three distinct breast cancer cell lines. The results show that the extent of H1 phosphorylation can distinguish between the different cell lines. The histone H1 from the metastatic cell line, MDA-MB-231, was subjected to chemical derivitization and LC-MS/MS analysis. The results suggest that the phosphorylation at threonine 146 is found on both histone H1.2 and histone H1.4. Cell lines were then treated with an extracellular stimulus, estradiol or kinase inhibitor LY294002, to monitor changes in histone H1 phosphorylation. The data show that histone H1 phosphorylation can increase and decrease in response to extracellular stimuli. Finally, primary breast tissues were stained for the histone H1 phosphorylation at threonine 146. Variable staining patterns across tumor grades and subtypes were observed with pT146 labeling correlating with tumor grade. These results establish the potential for histone H1 phosphorylation at threonine 146 as a clinical biomarker in breast cancer.

To the editor: Ibrutinib is an irreversible inhibitor of Bruton’s tyrosine kinase (BTK) with promising activity in CD20+ B-cell malignancies including recent US Food and Drug Administration approval in mantle cell lymphoma.[1][1] Given the homology between BTK and interleukin-2 inducible tyrosine

Introduction:T cell engaging bispecific antibodies (T-BsAb) redirect cytotoxic T cells to tumor associated antigen (TAA)-positive target cells, leading to T-cell activation and lysis of target cells. Blinatumomab is the only T-BsAb approved by FDA. Numerous T-BsAb are undergoing clinical trials, targeting both hematological malignancies (e.g. BCMA, CD123 and CD33) and solid tumors (e.g. CEA, EpCAM, and GPC3). While these T-BsAbs represent a promising novel therapeutic approach, their efficacy remains modest. Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and induces durable remissions in chronic lymphocytic leukemia (CLL). Our group has reported that ibrutinib has favorable immunomodulatory effects due to inhibition of interleukin-2 inducible T-cell kinase (ITK). Ibrutinib decreased key immunosuppressive checkpoint molecules and the frequency of regulatory T cells (Treg)(Long, Beckwith et al. 2017). Moreover, Ibrutinib significantly increased the number of the activated T cells by rescuing them from activation induced cell death (AICD). However, the function of ibrutinib 'rescued' T cells and their ability to mediate T-BsAb redirected cytotoxicity is unknown. Therefore we proceeded to study how ibrutinib treatment affects the functional competency of T cells by measuring their capability to mediate Blinatumomab redirected cytotoxicity. To examine T cell function post ibrutinib treatment, T cells were isolated from pre and post treatment PBMC samples by fluorescence activated cell sorting (FACS), and used as effector cells with pre-treatment CLL B-cells as target cells. Blinatumomab redirected T-cell cytotoxicity against tumor cells and T cell survival were assayed by flow cytometry based methods. Effector cells (T cells) were labeled with CFSE. Target (CLL) cells were labeled with CellTrace Violet. They were co-cultured at an E:T ratio of 4:1 with Blinatumomab for 24 hours. Effector cells and target cells can be distinguished by different fluorescence labels. Apoptosis and cell death were evaluated by Annexin-V and PI staining. The number of viable target cells and effector cells were calculated by Countbright counting bead. Results: We observed that post-ibrutinib T cells demonstrated significantly superior cytotoxicity against autologous CLL cells in the presence of blinatumomab in three independent patient samples. Numbers of viable CLL cells co-cultured with post-ibrutinib T cells were lower when compared to those co-cultured with pre-ibrutinib T cells (33.6%, 68% and 55.1% reduction in viable CLL cells for the three patient samples, respectively). We also found that Treg depletion (by depleting CD25high/CD127low CD4+ T cells) further enhanced cytotoxicity for both pre and post-ibrutinib treatment T cells, with the numbers of viable CLL cells reduced another 30-40%. Moreover, compared to pre-ibrutinib T cells, post-ibrutinib treatment T cells again demonstrated superior cytotoxicity when Treg cells were depleted, with decreased numbers of viable CLL cells at the end of co-culture (43%, 69% and 49.4% reduction, respectively). We also observed that the numbers of viable T cells from pre-ibrutinib samples were much lower than post-ibrutinib samples (52.5%, 77.9% and 74% reduction in viable T cells, respectively). Conclusions: Our findings suggest that ibrutinib-rescued T cells are functionally competent. CLL patients' T cells post ibrutinib treatment demonstrated superior cytotoxicity against autologous leukemia cells compared to T cells from treatment baseline. Moreover, we show that increased T cell activity is not solely due to ibrutinib-related Treg depletion. Treg depletion in our experiments further enhanced the cytotoxicity of both post and pre-ibrutinib treatment T cells while post-ibrutinib T cells still demonstrated superior cytotoxicity (Fig 1). Lastly, T cells isolated from post-ibrutinib samples demonstrated significantly improved viability after in-vitro stimulation with blinatumomab, which is likely a result of reduced AICD. Disclosures Bhat: Pharmacyclics: Consultancy; Janssen: Consultancy. Rogers:Janssen: Research Funding; AbbVie: Research Funding; Genentech: Research Funding; Acerta Pharma: Consultancy. Woyach:Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Verastem: Research Funding. Muthusamy:Ohio State University: Patents & Royalties: OSU-2S. Byrd:Novartis: Other: Travel Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Genentech: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; BeiGene: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau.

Several RNA-targeted therapeutics, including antisense oligonucleotides (ONs), small interfering RNAs, and miRNAs, constitute immunostimulatory CpG motifs as an integral part of their design. The limited success with free antisense ONs in hematologic malignancies in recent clinical trials has been attributed to the CpG motif–mediated, TLR-induced prosurvival effects and inefficient target modulation in desired cells. In an attempt to diminish their off-target prosurvival and proinflammatory effects and specific delivery, as a proof of principle, in the present study, we developed an Ab-targeted liposomal delivery strategy using a clinically relevant CD20 Ab (rituximab)–conjugated lipopolyplex nanoparticle (RIT-INP)– and Bcl-2–targeted antisense G3139 as archetypical antisense therapeutics. The adverse immunostimulatory responses were abrogated by selective B cell–targeted delivery and early endosomal compartmentalization of G3139-encapsulated RIT-INPs, resulting in reduced NF-κB activation, robust Bcl-2 down-regulation, and enhanced sensitivity to fludarabine-induced cytotoxicity. Furthermore, significant in vivo therapeutic efficacy was noted after RIT-INP–G3139 administration in a disseminated xenograft leukemia model. The results of the present study demonstrate that CD20-targeted delivery overcomes the immunostimulatory properties of CpG-containing ON therapeutics and improves efficient gene silencing and in vivo therapeutic efficacy for B-cell malignancies. The broader implications of similar approaches in overcoming immunostimulatory properties of RNA-directed therapeutics in hematologic malignancies are also discussed.

Chronic lymphocytic leukemia (CLL) cells are susceptible to oxidative stress. The expanded porphyrin, motexafin gadolinium (MGd), reacts with intracellular reducing metabolites and protein thiols to generate reactive oxygen species (ROS). A phase II trial administered MGd 5 mg/kg/day IV for 5 days every 3 weeks until disease progression to patients with previously treated CLL and small lymphocytic lymphoma. Thirteen patients (median age 66 years) with a median of four prior therapies (range 2-9) were enrolled. Modest anti-tumor activity was seen in three patients, with improvement in lymphocytosis, lymphadenopathy and/or splenomegaly, but no patient achieved a partial or complete response by NCI 96 criteria. Flow cytometry confirmed tumor uptake of MGd. Serial increase in AKT phosphorylation in patient samples following MGd treatment was not observed, suggesting intracellular generation of ROS was not optimal. Therefore, this schedule of administration achieved MGd uptake into primary tumor cells in vivo, but clinical activity was modest.

Interleukin-21 (IL-21) is a recently identified γ-chain receptor cytokine family member that promotes B-cell apoptosis as well as activation of innate immune system. Based on this, we hypothesized that IL-21 might enhance the apoptosis induced by fludarabine and rituximab and also play a role in augmenting immune-mediated clearance of the chronic lymphocytic leukemia (CLL) cells. Our studies demonstrate that the majority of CLL patients have surface IL-21 receptor-α, and its expression correlates with apoptosis, tyrosine phosphorylation of STAT1, and up-regulation of the proapoptotic BH3 domain protein BIM. IL-21–induced BIM up-regulation is critical for apoptosis because inhibition of BIM expression using small interfering RNA prevented IL-21–induced apoptosis. IL-21 treatment of CLL cells but not normal T cells with fludarabine or rituximab additively enhanced the direct cytotoxic effect of these therapies. In addition to its proapoptotic effect, IL-21 promoted STAT1 and STAT5 phosphorylation in natural killer cells with concurrent enhanced antibody-dependent cellular cytotoxicity against rituximab-coated CLL cells in vitro. These data provide justification for combination studies of IL-21 with fludarabine and rituximab in CLL and suggest that BIM up-regulation might serve as relevant pharmacodynamic end point to measure biologic effect of this cytokine in vivo.

Treatment options for chronic lymphocytic leukaemia (CLL) are limited and eventually fail because of the development of toxicities or drug resistance. Thus, identification of new therapeutic strategies and targets is a high priority. The semisynthetic geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) inhibits heat shock protein 90 (Hsp90) binding to client proteins, thereby leading to their degradation. We demonstrate that at biologically active and clinically attainable levels (1 mumol/l), 17-AAG treatment of CLL B cells in vitro causes modest apoptosis as well as decreased AKT protein levels. Given the potential activation of AKT following antibody therapy in CLL, we evaluated the combination of 17-AAG and rituximab. These agents produced synergistic cytotoxicity of CLL cells in vitro. However, rituximab-mediated antibody-dependent cellular cytotoxicity was modestly reduced with 17-AAG, and complement-dependent cytotoxicity was not altered. We conclude that the combination of Hsp90 inhibitors with therapeutic antibodies, such as rituximab may represent a novel strategy to enhance therapeutic response in CLL. Furthermore, our data indicates that AKT and Hsp70 protein levels are relevant pharmacodynamic endpoints to monitor the in vivo effect of 17-AAG therapy.

Purpose: The antiapoptotic Bcl-2 family member protein Mcl-1 is dynamically regulated in transformed B-cells, has a short mRNA and protein half-life, and is rapidly processed during apoptosis. Multiple therapies cause down-regulation of Mcl-1 in chronic and acute lymphoid leukemia (CLL and ALL) cells. Mcl-1 has also been reported to mediate resistance to rituximab in CLL. We therefore investigated whether direct reduction of Mcl-1 was sufficient to induce apoptosis and increase sensitivity to rituximab. Experimental Design: We used Mcl-1–specific small interfering RNA in ALL cell lines and tumor cells from CLL patients to block transcription of Mcl-1. Results: We show that Mcl-1 down-regulation alone is sufficient to promote mitochondrial membrane depolarization and apoptosis in ALL and CLL cells. Given the importance of rituximab in B-cell malignancies, we next assessed the influence of Mcl-1 down-regulation on antibody-mediated killing. Mcl-1 down-regulation by small interfering RNA increased sensitivity to rituximab-mediated killing both by direct apoptosis and complement-dependent cytotoxicity, but did not enhance antibody-dependent cellular cytotoxicity. Conclusions: These results show that Mcl-1 is a relevant therapeutic target for ALL and CLL, and its down-regulation has the potential to enhance the therapeutic effect of rituximab in CD20-bearing lymphoid cells.

Alemtuzumab is a humanized IgG1 kappa antibody directed against CD52, a glycosyl-phosphatidylinositol linked cell-membrane protein of unknown function. Herein, we demonstrate that alemtuzumab promotes rapid death of chronic lymphocytic leukemia (CLL) cells in vitro, in a complement and accessory cell free system. Using minimal detergent solubilization of CLL membranes, we found that CD52 colocalizes with ganglioside GM-1, a marker of membrane rafts. Fluorescence microscopy revealed that upon crosslinking CD52 with alemtuzumab+anti-Fc IgG, large patches, and in many cases caps, enriched in CD52 and GM-1 formed upon the CLL cell plasma membrane. Depletion of membrane cholesterol or inhibition of actin polymerization significantly diminished the formation of alemtuzumab-induced caps and reduced alemtuzumab-mediated CLL cell death. We compared alemtuzumab-induced direct cytotoxicity, effector cell-mediated toxicity and complement-mediated cytotoxicity of CLL cells to normal T cells. The direct cytotoxicity and observed capping was significantly greater for CLL cells as compared to normal T cells. Cell-mediated and complement-mediated cytotoxicity did not significantly differ between the two cell types. In summary, our data support the hypothesis that alemtuzumab can initiate CLL cell death by crosslinking CD52-enriched lipid rafts. Furthermore, the differential direct cytotoxic effect suggests that CD52 directed antibodies could possibly be engineered to more specifically target CLL cells.

The 1D10 antigen is the target for Hu1D10 (apolizumab), a humanized HLA-DR β-chain–specific antibody that is currently in clinical trials for hematologic malignancies. We demonstrate that Hu1D10 induces caspase-independent apoptosis following secondary cross-linking in primary chronic lymphocytic leukemia (CLL) cells. Generation of reactive oxygen species (ROS) and signal transduction, as evidenced by phosphorylation of Syk and AKT, were noted. The source of the Hu1D10-induced ROS was examined using the Raji lymphoblastic cell line with engineered defects in the mitochondrial respiratory chain. Hu1D10 treatment of clones with deficient mitochondrial respiration produced ROS suggesting a cytoplasmic source. Administration of ROS scavengers to primary CLL cells prior to Hu1D10 treatment diminished AKT activation. Treatment with Hu1D10 and the phosphatidylinositol 3-kinase inhibitor LY294002 demonstrated in vitro synergy with enhanced apoptosis. In conjunction with an ongoing clinical trial, blood samples were collected following intravenous infusion of Hu1D10 and analyzed for phosphorylation of AKT. Two of 3 patient samples showed a sustained increase in AKT phosphorylation following Hu1D10 administration. These data suggest that Hu1D10 ligation in CLL cells induces death and survival signals for which combination therapies may be designed to greatly enhance efficiency of both Hu1D10 and other class II antibodies in development.

XmAb-5574 antibody demonstrates superior antibody-dependent cellular cytotoxicity as compared with CD52- and CD20-targeted antibodies in adult acute lymphoblastic leukemia cells

Monocytes/macrophages are potent mediators of antitumor antibody therapy, where they engage target cells via Fcγ receptors (FcγR). Binding of these cells to opsonized tumor targets elicits cytokine production, phagocytosis, and antibody-mediated cellular cytotoxicity. Here we show for the first time that activation of monocyte FcγR results in the secretion of soluble vascular endothelial growth factor receptor-1 (VEGFR-1/sFlt-1), which serves to antagonize VEGF-mediated angiogenesis and tumor growth. Consistent with this, using a murine solid tumor model of antibody therapy, we show that sFlt-1 is involved in restricting tumor growth. Analyzing the mechanism of induction of sFlt-1, we found that the Erk and PI3K pathways were required for transcription, and NF-κB was required for translation. Upon closer examination of the role of NF-κB, we found that a microRNA, miR181a, negatively regulates FcγR-mediated sFlt-1 production and that NF-κB serves to antagonize this microRNA. Taken together, these results demonstrate a novel and biologically important function of monocytes and macrophages during antibody therapy.

Background: Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) that induces durable remissions in chronic lymphocytic leukemia (CLL). In other systems, ibrutinib has also been shown to have an immune modulating effect on T-cells through inhibition of interleukin-associated tyrosine kinase (ITK). Our group and then others have reported that ibrutinib enhances antibacterial and antitumor T cell immune response in murine models. ITK has been demonstrated to be involved in TCR-induced up-regulation of FAS Ligand (FAS-L) and activation induced cell death (AICD) of T cells. In murine models, ITK deficiency or inhibition by specific kinase inhibitors lead to impaired AICD and a much greater persistence and accumulation of activated T cells. We therefore postulate that ibrutinib, by its capability to irreversibly inhibit ITK, may have similar effect on recusing activated T cells from AICD. To date, a comprehensive analysis of the effects on cellular immunity in humans treated with ibrutinib, or more selective BTK inhibitors has not been reported. Methods: PBMCs from previously treated CLL patients enrolled on clinical trials (NCT01589302 and NCT02029443) of ibrutinib (a BTK and ITK inhibitor) and acalabrutinib (ACP-196, a selective BTK inhibitor) were collected at baseline and during treatment at the completion of 2 months( cycle 3) and 5 months (cycle 6). Patients with persistent lymphocytosis post treatment were chosen to allow comparable tumor burden before and after therapy. T cell immune function and the immunosuppressive capacity of CLL cells were evaluated with detailed immunologic and functional studies. Mechanistic studies examining AICD with ibrutinib and acalabrutinib treatment were performed. Results: A total of 25 patients treated with ibrutinib (n=17) and acalabrutinib (n=8) were studied. Ibrutinib, but not acalabrtinib treatment promoted a significant increase (3-5 fold) in absolute CD4(P Conclusions:These findings collectively differentiate ibrutinib as a therapeutic that can both expand activated T-cells and also ameliorate T-cell exhaustion thorough reversal of CLL tumor cell mediated immune suppression. While acalabrutinib reverses CLL cell mediated immune suppression, it lacks ability to expand activated T-cells. Collectively, these data provide support for dual BTK/ITK inhibitor therapeutics when applied to enhance the therapeutic efficacy of adoptive immunotherapy (e.g.chimeric antigen receptor T cells) or tumor vaccines for CLL and potentially other types of hematologic and solid cancers. Figure 1 Ibrutinib but not acalabrutinib treatment of CLL patients increases total T cell numbers: (A) Absolute numbers of CD8 T cells before and during ibrutinib treatment (n=18). (B) Absolute numbers of CD8 T cells before and during acalabrutinib treatment (n=8). Each cycle is 4 weeks. "Cycle 3" day 1 indicates 8 weeks into treatment, and "cycle 6" day 1 indicates 20 weeks into treatment. Figure 1. Ibrutinib but not acalabrutinib treatment of CLL patients increases total T cell numbers: (A) Absolute numbers of CD8 T cells before and during ibrutinib treatment (n=18). (B) Absolute numbers of CD8 T cells before and during acalabrutinib treatment (n=8). Each cycle is 4 weeks. "Cycle 3" day 1 indicates 8 weeks into treatment, and "cycle 6" day 1 indicates 20 weeks into treatment. Figure 2 Ibrutinib treatment of human T cells protects against activation induced cell death and reduces FAS-L mRNA expression in a dose dependent manor. T cells were isolated from healthy donors blood samples, stimulated in vitro with CD3/CD28 for 3 days, rested for 11 days, then were restimulated with CD3 for 6 hours (as in A. and B.) or 3 hours (as in C.) in the presence of IL2 to induce AICD in the presence or absence of ibrutinib. Figure 2. Ibrutinib treatment of human T cells protects against activation induced cell death and reduces FAS-L mRNA expression in a dose dependent manor. T cells were isolated from healthy donors blood samples, stimulated in vitro with CD3/CD28 for 3 days, rested for 11 days, then were restimulated with CD3 for 6 hours (as in A. and B.) or 3 hours (as in C.) in the presence of IL2 to induce AICD in the presence or absence of ibrutinib. Disclosures Jones: AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding. Andritsos:Hairy Cell Leukemia Foundation: Research Funding. Awan:Novartis Oncology: Consultancy; Innate Pharma: Research Funding; Pharmacyclics: Consultancy. Fraietta:Novartis: Patents & Royalties: Novartis, Research Funding. June:Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Tmunity: Equity Ownership, Other: Founder, stockholder ; University of Pennsylvania: Patents & Royalties; Immune Design: Consultancy, Equity Ownership; Pfizer: Honoraria; Johnson & Johnson: Research Funding; Celldex: Consultancy, Equity Ownership. Maus:Novartis: Patents & Royalties: inventor on patents related to ibrutinib use in T cell therapies. Woyach:Karyopharm: Research Funding; Morphosys: Research Funding; Acerta: Research Funding.

Introduction: Regulatory B cells (B-reg cells) and cytokine producing B cells have been increasingly recognized as key regulators of adaptive and innate immunity. Malignant B cells, such as chronic lymphocytic leukemia (CLL) cells share many features of B-reg cells and suppress host adaptive immunity via multiple mechanisms, including production of suppressive cytokines such as IL-10, surface expression of checkpoint molecules such as PD-L1 and CD200, as well as inducing expansion of regulatory T cells and Myeloid-derived Suppressor Cells (MDSC). Ibrutinib is an orally available irreversible BTK inhibitor that has been proved to be a safe and effective therapy for CLL. Recently we and others have demonstrated favorable cellular immunomodulatory effects of Ibrutinib through inhibition of an alternative target interleukin-2 induced T-cell kinase (ITK) that promotes Th1/Tc1 polarization. Herein, we explore the influence of Ibrutinib on the immune suppressive features and the B-reg phenotype associated with malignant B cells. Methods: PBMCs were collected from CLL patients treated with Ibrutinib at the time of pretreatment, cycle 3 day 1 (8 weeks into treatment) and cycle 6 day 1(20 weeks into treatment). Surface expression of checkpoint molecules and immunosuppressive capability of CLL cells were evaluated. For brief stimulation (“B10” condition), PBMCs were stimulated with Phorbol Myristate Acetate (PMA)/Ionomycin/Golgi-stop plus CpG for 5 hours. For prolonged stimulation (“B10-Pro” condition), PBMCs were stimulated with CpG plus CD40L for 48 hours, PMA / Ionomycin / Golgi-stop were added for final 5 hours. Results: CLL cells collected from patients treated with Ibrutinib in vivo were significantly impaired in their capability to make IL-10 after in vitro stimulation. On average, there is about a threefold reduction (P< 0.001) in the frequency of cells producing IL-10 by cycle 3, and a fivefold reduction (P< 0.001) by cycle 6. We have also shown that during the first two cycles of Ibrutinib, patients' plasma levels of IL-10 decreased. The impaired IL-10 production by CLL cells collected from post-Ibrutinib treatment patients is likely to due to an intrinsic change of the malignant B cells, rather than from acute inhibition of BTK; since resting these CLL cells in vitro for 48 hours (which allows for regeneration of BTK) did not lead to a recovery of IL-10 production. Interestingly, IFNγ production after in vitro re-stimulation is increased in CLL cells collected post Ibrutinib treatment. The analysis of potential immunosuppressive molecules revealed 3-5 fold reduction in surface expression of CD200 and BTLA(P Conclusion: CLL cells cause both global and tumor specific immunosuppression via multiple mechanism recapitulating regulatory B cells including production of IL-10, expression of checkpoint molecules such as CD200, and induction of suppressive/regulatory cells such as Treg cells and MDSC cells. Here we demonstrate that Ibrutinib treatment has a powerful in vivo immunomodulatory effect on the malignant B cells by counteracting all the aforementioned B-reg like activities, which makes it a promising immunotherapeutic agent. Note: This abstract was not presented at the conference. Citation Format: Meixiao Long, Kyle A. Beckwith, Priscilla Do, Amber Gordon, Amy Lehman, Kami Maddocks, Carolyn Cheney, Jeffrey Jones, Leslie Andritsos, Farrukh Awan, Jennifer Woyach, Johnson J. Amy, Natarajan Muthusamy, John Byrd. Ibrutinib treatment counteracts the immunosuppressive activity of malignant B cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B041.

7530Background: BTK is an attractive target in CLL. Ibrutinib (IB), a first in class irreversible BTK inhibitor (BTKi), abrogates important survival signals in CLL by proximally blocking multiple p...

Apolizumab (Hu1D10), a humanized monoclonal anti- Human leukocyte antigen -DR beta-chain antibody, mediates apoptosis of chronic lymphocytic leukemia (CLL) cells in vitro. We conducted a phase I/II dose-escalation study of thrice-weekly apolizumab (1.5, 3.0, 5.0 mg/kg/dose) for 4 weeks in relapsed CLL. Two of six patients at 5.0 mg/kg/dose developed treatment-related dose-limiting toxicity (aseptic meningitis, hemolytic uremia). Other toxicities included infusion toxicity, urticaria, and headache. Eleven patients were enrolled in a phase I/II expansion to evaluate the maximum tolerated dose (MTD) of 3.0 mg/kg/dose. In total, 23 patients were enrolled (22 CLL, 1 ALL). Nineteen patients with CLL were treated at or above the MTD. One partial response was observed, and three patients had stable disease exceeding 6 months. Pharmacokinetic analysis demonstrated a dose-dependent C(max) increase and serum antibody accumulation after week 1 of therapy. Given the toxicity and lack of efficacy in this and other trials in lymphoma and solid tumors, further development of apolizumab was discontinued.

Purpose: To date, efforts to study CD52-targeted therapies, such as alemtuzumab, have been limited due to the lack of stable CD52 expressing transformed B-cell lines and animal models. We describe generation and utilization of cell lines that stably express CD52 both in vitro and in vivo.Experimental Design: By limiting dilution, we have established several clones of Raji-Burkitt's lymphoma cell line that express surface CD52. Immunophenotype and cytogenetic characterization of these clones was done. In vivo usefulness of the CD52high cell line to evaluate the therapeutic efficacy of CD52-directed antibody was investigated using a SCID mouse xenograft model.Results: Stable expression of CD52 was confirmed in cells cultured in vitro up to 52 weeks of continuous growth. The functional integrity of the expressed CD52 molecule was shown using alemtuzumab, which induced cytotoxic effects in vitro in the CD52high but not the CD52low clone. Compared with control antibody, alemtuzumab treatment in CD52high inoculated mice resulted in significantly increased median survival. Comparable levels of CD52-targeted direct cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cytotoxicity and anti-CD52 immunoliposome-mediated delivery of synthetic oligodeoxyribo nucleotides in CD52high clone and primary B-chronic lymphocytic leukemia cells implicated potential in vivo application of this model for evaluation of CD52-targeted antibody and immunoliposomes encapsulating therapeutic agents.Conclusions: These results show the in vitro utility of the cloned Raji cell lines that stably express high levels CD52. The disseminated leukemia-lymphoma mouse model described herein using these stable cell lines can serve as an excellent system for in vivo therapeutic and mechanistic evaluation of existing and novel antibodies directed against CD52 molecule.

Lumiliximab is a chimeric macaque-human monoclonal antibody to CD23, a protein expressed on virtually all chronic lymphocytic leukemia (CLL) cells. We examined the ability of lumiliximab to mediate apoptosis, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity against primary CLL cells and CD23-expressing B-cell lines. Our data suggest that lumiliximab kills CLL cells and CD23-expressing B cells predominantly by apoptosis, which occurs through the intrinsic pathway. Lumiliximab-induced apoptosis was accompanied by the down-regulation of antiapoptotic proteins Bcl-2, Bcl-XL, and XIAP, activation of Bax, and release of cytochrome c from the mitochondria. We also found that the addition of lumiliximab to rituximab or fludarabine results in synergistic cytotoxicity of primary CLL cells and CD23-expressing B-cell lines. We investigated the in vivo activity of lumiliximab in a human disseminated CD23+ B-cell lymphoma SCID mouse model and found greater antitumor activity with it than with control antibody. We also found that paralysis-free survival was greater with lumiliximab plus rituximab or fludarabine than with any of those agents alone. These results suggest that lumiliximab may be an effective treatment alone or in combination with rituximab or chemotherapy agents in CLL or other CD23-overexpressing B-cell malignancies.

Novel chronic lymphocytic leukemia (CLL) therapies target phosphoinositide 3-kinase (PI3K; idelalisib) and Bruton's tyrosine kinase (BTK; ibrutinib). Despite their success, certain high-risk groups, notably those with dysfunctional P53, demonstrate lower response rates than other CLL groups. As such, combination therapies could potentially overcome this deficiency. As CD37-mediated signaling resulted in direct cytotoxicity that was enhanced with PI3K inhibition (Lapalombella 2012), we aimed to combine BI 836826, a novel IgG1 chimerized and Fc-engineered anti-CD37 mAb, with BCR-pathway inhibitors to possibly enhance clinical efficacy in high-risk CLL patients and overcome potential inhibition of ADCC. Consistent with previous report, BI 836826 displays both ADCC and direct pro-apoptotic activity (Heider 2011). We assessed the potential effect of idelalisib and ibrutinib on CD37 expression and NK-cell function. Following 24hr incubation of primary CLL cells (n=4) with idelalisib, ibrutinib, or DMSO, there was no significant difference in surface expression of CD37 (fold change ~ 1.01; p > 0.16 for all comparisons). To ensure that NK-cell Fc-receptor function was unimpaired by BCR-pathway inhibitors, we conducted CD107ab degranulation assays (n=5). Data revealed that Fc-receptor engagement of BI 836826 and NK-cell lytic degranulation was not abrogated by idelalisib or ibrutinib (mean CD107ab expression: DMSO = 10.14%; idelalisib = 5.67%; ibrutinib = 2.41%). In combination with idelalisib, BI 836826 was still capable of eliciting an effective NK-cell response superior to rituximab (difference = 4.51%; p < 0.0001). To further qualify NK-cell function, TNFα (n = 6) or INFγ (n = 12) release by NK cells after pretreatment with idelalisib or ibrutinib was evaluated. Similarly, cytokine release was not completely inhibited by idelalisib and ibrutinib; and BI 836826 was still capable of inducing cytokine release. To confirm the potential added benefit of combination therapy, we conducted 51Cr release ADCC assays with primary CLL cells and healthy allogeneic NK-cells (Fig1) or CLL patient autologous NK-cells (Fig2). Results confirmed that neither ibrutinib nor idelalisib could ablate BI 836826-induced NK-cell ADCC. However, there was a more pronounced inhibition of NK-cell ADCC with ibrutinib. As previously demonstrated (Kohrt 2014), NK-cell ADCC mediated by rituximab was almost completely abrogated by ibrutinib, likely secondary to off-target interleukin-2-inducible T-cell kinase inhibition (Dubovsky 2013). Our preliminary data show that signaling induced by BI 836826 may demonstrate the same enhanced direct cytotoxicity with PI3K inhibition as previously reported with other CD37 mediated signaling agents (Lapalombella 2012). Evaluation of direct pro-apoptotic activity of BI 836826 (0.1ug/mL) in combination with idelalisib (1uM) on direct cell death (24hr incubation) demonstrated enhanced activity in the combination setting with similar activity in primary patient CLL cells with dysfunctional P53 (n=6; Fig3A) and functional P53 (n=4; Fig3B). In conclusion, these results demonstrate that idelalisib could potentially be used in combination with BI 836826, which has the added benefit of directly killing inhibitor-resistant P53-null primary CLL cells and lacks complete inhibitor-induced ADCC ablation. Figure 1. Allogeneic ADCC after Pretreatment of NK-cells with BTK inhibitor (Ibrutinib) or PI3K inhibitor (Idelalisib) Figure 1. Allogeneic ADCC after Pretreatment of NK-cells with BTK inhibitor (Ibrutinib) or PI3K inhibitor (Idelalisib) Figure 2. Autologous ADCC after Pretreatment of NK-cells with BTK inhibitor (Ibrutinib) or PI3K inhibitor (Idelalisib) Figure 2. Autologous ADCC after Pretreatment of NK-cells with BTK inhibitor (Ibrutinib) or PI3K inhibitor (Idelalisib) Figure 3. BI 836826 Direct Cytotoxicity in High- and Low-Risk CLL Patients Figure 3. BI 836826 Direct Cytotoxicity in High- and Low-Risk CLL Patients Disclosures Off Label Use: Off-label use of idelalisib and BI 836826 in combination for CLL patients. Jones:Pharmacyclics: Consultancy, Research Funding. Heider:Boehringer Ingelheim: Employment.

Abstract 1847 CD37 is a tetraspanin transmembrane family protein that is strongly expressed on the surface of mature human B cells and transformed mature B cell lymphoma and leukemia cells, including CLL cells. It is absent or minimally expressed on normal T cells, NK cells, monocytes, and granulocytes. Predominant expression of CD37 on CLL cells makes it an ideal candidate to target with potential agents for treatment of CLL. TRU-016, a small modular immunopharmaceutical protein (SMIP) that specifically binds to an extracellular region of CD37, is presently in clinical trials in CLL patients. TRU-016 includes humanized immunoglobulin variable regions (scFv) fused to a human IgG1 Fc region. We have previously reported that SMIP-016, the chimeric version of the humanized TRU-016, induced apoptosis in CLL B cells in the presence of goat anti-human Fc antibody cross-linker through a novel, caspase-independent pathway. Furthermore, SMIP-016 showed potent in vivo activity in a SCID xenograft mouse model. Aside from direct cytotoxicity, SMIP-016 mediates antibody-dependent cellular cytotoxicity (ADCC) by NK cells both in vitro and in vivo. In an attempt to enhance its ADCC function, a new variant of SMIP-016, SMIP-016GV, was designed with a modification of the glycosylation of the Fc portion of the molecule. SMIP-016GV exhibits enhanced binding to both low- and high-affinity molecular variants of human CD16 (FcγRIII) and augments ADCC potency when compared to SMIP-016. In this study, we compared SMIP-016GV and SMIP-016 in direct cytotoxicity and ADCC against CLL B cells. While SMIP-016 and SMIP-016GV mediated comparable direct cytotoxicity at 48 hrs in the presence of goat anti-human Fc crosslinker, the SMIP-016GV resulted in 2 to 4 fold increase in NK cell mediated ADCC function at all effector to target ratios tested. This increased ADCC with SMIP-016GV was observed using NK cell effectors derived from both normal as well as CLL-affected individuals. In addition, this enhanced cytotoxicity was sustained at concentrations of SMIP-016GV as low at 5E-6 μg/ml. These low concentrations of SMIP-016GV were also able to mediate superior ADCC in 697 cells expressing as few as 10,000 molecules of surface CD37 antigen. Furthermore, NK cells stimulated with the glycovariant were potently activated and released 3 to 4 fold more IFNγ compared to SMIP-016. Ongoing studies are aimed at defining other effector cells which may interact with SMIP-016GV via different Fcγ Receptors. Collectively, these results suggest potential use of the SMIP-016GV with enhanced ADCC function as an alternate for TRU-016 in B cell malignancies including CLL therapy. Disclosures: Siadak: Trubion Pharmaceuticals: Employment.

Abstract 1744 Poster Board I-770 CD37 is a tetraspanin transmembrane family protein that is strongly expressed on the surface of mature human B-cells and transformed mature B-cell lymphoma and leukemia cells, including CLL cells. It is expressed minimally or is absent on normal T-cells, natural killer cells, monocytes, and granulocytes. Predominant expression of CD37 on CLL cells makes it an ideal candidate to target with potential agents for treatment of CLL. TRU-016, a Small Modular ImmunoPharmaceutical protein (SMIP) targeted towards the extracellular region of CD37, is presently in clinical trials in CLL patients. TRU-016 consists of variable regions (scFv) and engineered constant regions encoding the human IgG1 domains. We have previously reported that SMIP-016, the chimeric precursor of the fully humanized TRU-016, induced apoptosis in CLL B cells in the presence of goat anti-human Fc ab cross-linker through a novel, caspase-independent pathway. Furthermore, SMIP-016 showed potent in-vivo activity in a SCID xenograft mouse model. Aside from direct cytotoxicity, SMIP-016 mediates antibody-dependent cellular cytotoxicity (ADCC) by NK cells both in vitro and in vivo. Recently, in an attempt to enhance the ADCC function, a new variant of SMIP-016, Tru-ADhanCe SMIP-016, has been created with a modification of the glycosylation of the Fc portion of the molecule. TRU-ADhanCe SMIP-016 has been shown to exhibit enhanced binding to both low- and high-affinity molecular variants of human CD16 (FcRIII) and augmented ADCC potency when compared to SMIP-016. In this study, we compared TruADhanCe SMIP-016 and SMIP-016 in direct cytotoxicity and ADCC experiments using CLL B-cells. While SMIP-016, and TruADhanCe SMIP-016 mediated comparable direct cytotoxicity at 24, 48 and 72 hrs in the presence of anti-human Fc crosslinker, the TruADhanCe SMIP-016 resulted in 2 to 4 fold increased NK cell mediated ADCC function. Consistent with the comparable direct cytotoxic effects, the early phosphorylation patterns were similar in cells treated with TruADhanCe SMIP-016 or SMIP-016 in the presence of anti-human Fc cross linker. Ongoing studies are aimed to define the mechanistic basis of the enhanced ADCC function by TruADhanCe SMIP-016 and to determine if use of soluble CD16.Fc as a cross-linker, an in vitro model of in vivo Fc receptor binding, may reveal enhanced apoptotic-signaling of TruADhanCe SMIP-016. These results suggest potential use of TruADhanCe versions of TRU-016 with enhanced ADCC function as an alternate for TRU-016 in B cell malignancies including CLL therapy. [This work was supported by D. Warren Brown Foundation, Leukemia and Lymphoma Society and National Cancer Institute.] Disclosures Thompson: Trubion Pharmaceuticals: Employment. Siadak:Trubion Pharmaceuticals: Employment. Algate:Trubion Pharmaceuticals: Employment. Cerveny:Trubion Pharmaceuticals: Employment.

7094 Chemoimmunotherapy with fludarabine and rituximab has demonstrated promising clinical activity in CLL. We hypothesized that the IL-21 might enhance the efficacy of this regimen and play a role in immune mediated clearance of CLL cells. Expression of IL21R-a was present (>20% cells +) in 13 of 16 (81%) pts. IL-21 induced direct cytotoxicity of CLL cells (>20% apoptosis) in subset of patients (n=9, 40% ± 15.9, ptyr- 701 and STAT-3tyr-705 was seen in cells with apoptosis >20% whereas phosphorylation of STAT-1tyr-701 was not seen in cells failing to undergo apoptosis. In contrast to this, responder and non responder cells exhibited comparable levels of STAT-1, STAT-3 and constitutive Ser727 phosphorylated STAT-3 levels. Further, IL-21 induced upregulation of Bim was observed in the cells undergoing apoptosis. Interestingly, IL-21 enhanced additively, the direct apoptosis by either antibody (rituximab or alemtuzumab) or fludarabine therapy (Pretreatment of CLL cells with IL-21 for 18–20 hours). In contrast to the enhanced fludarabine mediated direct cytotoxic death of B cells, IL-21 failed to enhance the fludarabine mediated death of T cells (n=5, % alive 56±15 vs 58±16 with IL-21) suggesting that this immunotherapeutic strategy will not augment the immunosuppressive effect of fludarabine. IL-21 also enhanced innate immune activation as demonstrated by killer inhibitory receptor matched autologous natural killer cell mediated antibody dependent cellular cytotoxicity against rituximab coated CLL cells by 33% (E: T ratio 25:1, 42% ± 4.4 versus 28% ± 3.1, p No significant financial relationships to disclose.

CD37 is a lineage-specific B-cell antigen that represents an attractive target for immunotherapy in B-cell malignancies, especially in chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL). CD37-specific small modular immuno pharmaceutical (CD37-SMIP™) drug is an engineered protein therapeutic directed to the CD37 antigen using a single chain variable region (scFv) linked to a modified human IgG1 hinge, CH2, and CH3 domains. We have previously presented that CD37-SMIP™ drug induces both ADCC and apoptosis against primary CLL cells and B-cell lymphoma cells and therapeutic efficacy was observed in a Raji cell disseminated leukemia xenograft model. Herein, we sought to determine the effector cell type(s) mediating ADCC and explore if agents that deplete NK cell inhibitory T-regulatory cells influence CD37-SMIP™ efficacy. Immunostaining of human peripheral blood mononuclear cells (PBMCs) from CLL patients demonstrated no expression of CD37 on CD3+ T cells, CD16+ or CD56+ NK cells, or CD64+ monocytes whereas CD37 was highly expressed on CD19+ B cells. Using purified human PBMCs as effector cells and Cr-51 labelled CLL B cells as targets, we found the CD37-SMIP™ dependent ADCC was predominantly mediated by NK cells, but not naïve or activated monocytes. Consistent with these in-vitro results, the in-vivo therapeutic efficacy of CD37-SMIP™ drug was significantly compromised by depletion of NK cells in the Raji cell disseminated leukemia xenograft SCID mouse model. The median survival time of CD37-SMIP™ treated mice decreased from 51 days (95% CI: 38, 78) to 27 days (95% CI: 25, 37) (p=0.017) with the depletion of NK cells. Consistent with previous studies, ADCC is not diminished by fludarabine, an agent that may deplete T-regulatory cells, suggesting that the anti-CD37 protein and fludarabine might combine for increased efficacy in-vivo. We therefore examined the effect of fludarabine on CD37-SMIP™ in-vitro apoptotic activity. These data demonstrate that direct cell death mediated by CD37-SMIP™ drug (5 ug/mL) synergizes with fludarabine-induced caspase-dependent apoptosis, as measured by both the MTT assay and annexin V/PI staining of CLL cells (combination index, CI=0.44). Overall, these data suggest that the CD37-SMIP™ is a promising therapeutic agent against CD37+ B-cell malignancies as either monotherapy or in combination with fludarabine. Further clinical development is warranted to investigate the effect of CD37-SMIP™ drug in CLL and NHL. (SMIP trademark is owned by Trubion Pharmaceuticals).

The direct effect of the feline leukaemia virus (FeLV) on erythroid colony formation in vitro was investigated. Bone marrow mononuclear cells (BMMC) from FeLV-naive, specificpathogen-free (SPF), adult cats were inoculated with FeLVs of characterized strains and biologically cloned subgroups and the subsequent development of colony forming units-erythroid (CFU E ) and burst forming units-erythroid (BFU E ) and colony forming units-granulocyte-macrophage (CFU GM ) was monitored. Exposure to the anaemia-causing Kawakami-Theilen strain of FeLV (FeLV-KT), a phenotypic mixture of subgroups A, B, and C, caused constant depression of day 2 CFU E (to 47% of sham-inoculated controls), day 4 CFU E (41% of controls), and day 10 BFU E (38% of controls). CFU GM were unaffected. The lymphoma-causing Rickard strain of FeLV (FeLV-R-TL) caused sporadic depression of CFU E and BFU E . In contrast, neither FeLV-R passaged through feline embryonic kidney fibroblasts (FeLV-R-CRFK) nor biologically cloned, subgroup-specific, FeLVs of fibroblast origin, caused decrements in CFU E or BFU E , suggesting that fibroblast passage attenuated the direct erythrosuppressive effect of FeLV. Suppression of CFU E and BFU E by lymphoma cell-origin FeLV was dependent on infectious virus and was associated with FeLV replication by the cultured myelomonocytic precursor cells. Attenuation of infectivity by heat or u.v. restored CFU E and BFU E development. Examination of the relationship between viral infectivity (VI), viral protein concentration, and CFU E suppression showed that the infectious FeLV was 20-fold more effective than u.v.-inactivated FeLV as an inhibitor of erythrogenesis in vitro .