TNT009 Prevents Erythrocyte C3 Fragment Opsonization and Rescues Reticulocytes from Destruction in Patients with Cold Agglutinin Disease

Abstract Autoantibody mediated classical complement pathway (CP) activation has been hypothesized to drive hemolytic anemia in cold agglutinin disease (CAD) patients. Red blood cell (RBC) destruction is believed to occur as a result of C3 opsonin mediated extravascular hemolysis in the liver. We recently reported that TNT009, a humanized monoclonal antibody targeting the CP specific serine protease C1s, rapidly restores hemoglobin levels in severely anemic CAD patients. Here we describe the pharmacodynamic changes in the complement profile of patients to provide a mechanistic understanding of the hematological responses and therapeutic benefit observed following TNT009 treatment. In a Phase 1b trial, we enrolled 5 female CAD patients with severe anemia, one of whom had a lymphoplasmacytic lymphoma with >70% bone marrow infiltration and no measurable CP activity prior to dosing. This patient did not respond to TNT009 while on study and will be omitted from subsequent analyses. Patients were given an initial 10 mg/kg test dose of TNT009 on Day 1, followed by four weekly doses of 60 mg/kg on Day 2 or Day 5. Patients were followed for 4 weeks following the last dose (washout). Plasma and serum samples were collected throughout the study to measure TNT009 concentrations and to monitor serological markers of anemia and hemolysis. Additionally, futhan-containing plasma samples were collected to assess the levels of CP specific components including C1s, C1s-C1INH, and C1q by ELISA. RBCs were collected to monitor cell surface complement deposition (C3 fragments) via flow cytometry. Finally, C4 levels and an ELISA-based readout of CP activity were examined as measures of the pharmacodynamic effect of TNT009. Baseline levels of circulating C4 were either low or undetectable in CAD patients. Accordingly, serum CP activity was reduced compared to normal human serum samples. Following the first 60 mg/kg TNT009 dose, CP activity was immediately and completely inhibited within 15 minutes of dosing in all patients and remained inhibited for 3 weeks after the last dose. During this period of inhibition, C4 levels rose from a median circulating concentration of <90 mcg/mL (range: <70 - 145) to 251 mcg/mL (range: 238 - 353; p < .001). Plasma C1s levels, on the other hand, decreased from a median plasma concentration of 53.3 mcg/mL (range: 49.4 - 60.3) to a nadir of <3.13 mcg/mL, the lower limit of quantification (LLOQ), (p < .001). Similarly, C1s-C1INH decreased from a median value of 4.5 mcg/mL (range: 4.2 - 5.3) to a nadir of <0.16 mcg/mL (LLOQ; p < .001). Notably, circulating plasma C1q levels were unaffected. Classical pathway inhibition led to a significant increase in reticulocytes in all patients by, on average, 69% within 24 hours of dosing (p < .05). Interestingly, within 1 week after the first TNT009 dose, reticulocyte counts returned to pre-treatment levels and continued to decrease throughout the study, as expected when hemoglobin normalizes. Similarly, within 24 hours of the first dose of TNT009, bilirubin levels dropped from a median value of 2.1 mg/dL (range 1.6 - 3.8) to 0.7 mg/dL (range 0.6 - 1.2), resulting in an average reduction of 66% from baseline levels (p < .05) and returning to pre-treatment levels following washout. Finally, we monitored in vivo complement activation by staining for C3 fragment deposition on RBCs. In general, we observed a gradual reduction in the percentage of C3 fragment positive RBCs over the course of the study from a median value of 49% (range: 37 - 81) to 29% (range: 20 - 38) before washout of TNT009. The decrease in opsonized RBCs was concomitant with the rise in hemoglobin (example shown in Figure 1). Figure 1: Elevation of hemoglobin is associated with a decrease in C3 fragment coated erythrocytes Here we report that TNT009 administration depletes circulating C1s and immediately halts in vivo CP activity, normalizing plasma C4 levels in CAD patients. The abrupt increase in reticulocyte count within 24 hours of dosing suggests that cold agglutinin mediated complement activation affects reticulocyte survival, preventing their maturation into erythrocytes. The observed reduction in C3 opsonized RBCs suggests that TNT009 ameliorates anemia by preventing complement mediated hepatic RBC sequestration, supported by the immediate normalization of circulating bilirubin levels. These results provide a mechanistic interpretation of the therapeutic effects of TNT009 in CAD patients. Figure 1. Figure 1. Disclosures Panicker: True North Therapeutics, Inc.: Employment, Equity Ownership. Hussain:True North Therapeutics, Inc.: Employment, Equity Ownership. Parry:Truenorth Therapeutics, Inc.: Employment, Equity Ownership. Gilbert:Truenorth Therapeutics, Inc.: Employment, Equity Ownership. Jaeger:Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses.

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Therapeutic Rationale and Clinical Development of TNT009, an Upstream Classical Pathway Inhibitor, for Cold Agglutinin Disease

Blood ◽

10.1182/blood.v126.23.3560.3560 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3560-3560 ◽

Cited By ~ 1

Author(s):

Ulrich Jaeger ◽

Eileen L Rose ◽

Andrew Singh ◽

Sebastian H A Feickert ◽

Simon Panzer ◽

...

Keyword(s):

Complement Activation ◽

Research Funding ◽

Cold Agglutinin ◽

Serum Samples ◽

Employment Equity ◽

Equity Ownership ◽

Human Rbcs ◽

D Dimer

Abstract Cold agglutinin disease (CAD) is an autoimmune hemolytic anemia (AIHA) characterized by the presence of autoantibodies (cold agglutinins) that bind red blood cells (RBCs) and activate the classical complement pathway (CP). We have previously shown in vitro that in contrast to C5 inhibition, inhibition of the CP specific protease C1s prevents complement opsonin deposition on cold agglutinin-sensitized RBCs and protects them from phagocytosis, underscoring the necessity to block upstream CP activity (Shi et al., Blood, 2014). Based on the strong scientific rationale and nonclinical data, a Phase 1 clinical trial for TNT009, a monoclonal antibody (mAb) inhibitor of C1s, has commenced at the Medical University of Vienna, Austria. Phase 1a consists of healthy volunteer cohorts in single- and multiple-ascending dose protocols for which interim study results will be presented. In the integrated protocol design of Phase 1b, TNT009 will be dosed in patients with diseases in which pathological CP activity has been implicated, including CAD, warm AIHA and additional non-hematologic indications. In anticipation of the clinical trial, we initiated a screening campaign in Vienna to find prospective CAD patients with serological markers of anemia and hemolysis. To date, plasma and serum samples have been collected from 15 CAD patients. Serum samples from 10 patients induce robust complement activation (C3b/iC3b deposition and/or hemolysis) on AET-treated human RBCs incubated in the patient's own complement-containing serum. In contrast to isotype control (IC), 100 mcg/mL of TNT003 (mouse parental mAb of TNT009), showed near complete inhibition of patient serum mediated C3b/iC3b deposition (90 ± 4%, n = 10; p< 1 x 10-5) and hemolysis (93 ± 5 %, n = 9; p< 1 x 10-5) (Fig. 1). To further support the rationale of C1s inhibition in CAD, we asked whether serological signs of anemia and hemolysis were associated with evidence of increased in vivo CP activity in patient samples. We first examined how well experimental laboratory results agreed with standard clinical readouts. We found good concordance between patient sample induced C3 deposition on RBCs (FACS) and clinical C3 DAT scores (p< .05). Furthermore, IgM staining on RBCs incubated in patient samples (FACS) correlated well with cold agglutinin titers determined in the clinic (p < .001). Next, we observed that the extent of in vitro hemolysis correlated with C3d DAT scores (p< .05), LDH levels (p< .05), and bilirubin levels (p= .05). The agreement between the results from our in vitro patient sample-induced hemolysis assay with serologicalmarkers of complement activity, hemolysis and anemia used in the clinic suggest that our in vitro paradigm serves as a good model for in vivo complement activity in CAD patients. We then measured plasma C4 levels and CP activity in CAD serum samples (Wieslab Classical Pathway ELISA). We found that plasma C4 positively correlated with hemoglobin levels (p = .05). Additionally, we found an inverse correlation between serum CP activity and reticulocyte count (p < .05) and bilirubin levels (p = .05). These data demonstrate that in vivo consumption of the CP and its components (low CP activity, low C4) is associated with markers of anemia and hemolysis (low hemoglobin, high reticulocyte counts, high bilirubin). Finally, an emerging literature calls attention to an increased thromboembolic risk in AIHA, similar to that seen in patients with other hemolytic anemias such as paroxysmal nocturnal hemoglobinuria. We therefore measured D-dimer levels and found it significantly elevated in CAD patient plasma compared to healthy controls (p < .0001). Preliminary analyses show an inverse correlation of C4 and D-dimer in patient plasma (p < .05) suggesting that in vivo CP activity may contribute to the elevated thromboembolic risk in patients (Fig. 2). On-going analyses for other markers of thrombosis, in addition to other experimental approaches to assess the hypercoagulable state in these patients will seek to corroborate this finding. The successful identification of CAD patients with altered complement and hematological profiles provides a unique opportunity to assess proof-of-concept early in the clinical development of TNT009. Figure 1. TNT009 Parental mAb (TNT003) Inhibits CAD Serum Mediated Complement Activation on AET-Treated Human RBCs Figure 1. TNT009 Parental mAb (TNT003) Inhibits CAD Serum Mediated Complement Activation on AET-Treated Human RBCs Figure 2. Elevated D-dimer Correlates with Lower C4 Levels in CAD Patient Plasma Figure 2. Elevated D-dimer Correlates with Lower C4 Levels in CAD Patient Plasma Disclosures Jaeger: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; True North Therapeutics, Inc.: Research Funding; Hoffmann La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rose:True North Therapeutics, Inc.: Employment, Equity Ownership. Singh:True North Therapeutics, Inc.: Employment, Equity Ownership. Jilma:True North Therapeutics, Inc.: Consultancy, Research Funding. Gilbert:True North Therapeutics, Inc.: Employment, Equity Ownership. Panicker:True North Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

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Phase II Study of the Cyclin-Dependent Kinase (CDK) Inhibitor Dinaciclib (SCH 727965) In Patients with Advanced Acute Leukemias

Blood ◽

10.1182/blood.v116.21.3287.3287 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3287-3287 ◽

Cited By ~ 5

Author(s):

Ivana Gojo ◽

Alison Walker ◽

Maureen Cooper ◽

Eric J Feldman ◽

Swaminathan Padmanabhan ◽

...

Keyword(s):

Research Funding ◽

Post Treatment ◽

Parp Cleavage ◽

Employment Equity ◽

Acute Leukemias ◽

Early Management ◽

Tumor Lysis ◽

Equity Ownership ◽

Pre Treatment

Abstract Abstract 3287 Background: Dinaciclib is a potent and selective inhibitor of the CDKs 1, 2, 5, and 9 that has demonstrated anti-tumor activity against both myeloid and lymphoid leukemia cell lines in vitro and human tumor xenografts in vivo. Methods: A randomized, multicenter, open-label phase 2 study of dinaciclib 50 mg/m2 administered by 2-hour i.v. infusion once every 21 days was initiated with the goal of assessing its efficacy and safety in patients (pts) with advanced acute myeloid (AML, ≥60 years old) or lymphoid (ALL, ≥18 years old) leukemia. AML pts were randomized between dinaciclib and gemtuzumab ozogamicin (GO) with cross-over to dinaciclib if no response to GO, while ALL pts only received dinaciclib. Intra-patient dose escalation of dinaciclib to 70 mg/m2 in cycle 2 was allowed. Twenty-six pts were treated on study (20 AML, 6 ALL). Data on 14 AML (2 cross-over from GO) and 6 ALL pts treated with dinaciclib are presented. Their median age was 70 (range 38–76) years and 70% were male. Sixteen pts were refractory and 4 pts had relapsed after a median of one (range 1–4) chemotherapy regimens. Four AML pts had complex karyotypes (≥3 abnormalities), 2 monosomy 7, 2 trisomy 8, 1 der (1:7)(q10;p10), 1 trisomy 21, 1 deletion 9q, and 3 had normal karyotype. Two ALL pts had t(9;22). Response: Anti-leukemia activity was observed in 60% of pts. Ten of 13 pts with circulating blasts (7/7 AML and 3/6 ALL) had >50% and 6 pts (4 AML, 2 ALL) >80% decrease in the absolute blast count (ABC) within 24 hours of the first dinaciclib dose. An additional pt had a 29% decrease in ABC. The median pre-treatment ABC was 1085 (range 220–9975) and the median ABC nadir was 169 (range 0–1350). The median duration of blast nadir was 6 days (range 2–23). A representative graph from an AML patient (below) shows a rapid decrease of circulating blasts and WBC after treatment, followed by a gradual recovery. Two patients had >50% reduction of marrow blasts (35% on d1 to 17% on d 42 in an AML pt; 81% on d1 to 27% on d 21 in an ALL pt). However, no objective responses by International Working Group criteria were observed. The median number of treatment cycles was 1 (range 1–5), with 10 pts receiving more than one cycle of treatment. Eight pts were treated with dinaciclib 70 mg/m2 starting in cycle 2. Toxicity: Treatment related AE's occurring in >30% of pts included diarrhea, nausea, vomiting, anemia, elevated AST, fatigue, leukopenia, hypocalcemia, and hypotension. The most common CTCAE v3 treatment-related grade 3 and 4 toxicities, occurring in 3 or more pts, were anemia, leukopenia, febrile neutropenia, thrombocytopenia, fatigue, increased AST, and tumor lysis syndrome (TLS). Laboratory evidence of tumor lysis in cycle 1, using the Cairo-Bishop criteria, was seen in 6 pts in addition to 3 pts with clinical TLS (JCO 2008;26:2767). Hyperacute TLS requiring hemodialysis occurred in one pt with AML, who died of acute renal failure. Subsequently, all pts were aggressively managed to prevent and treat TLS (hospitalization, hydration, allopurinol, rasburicase, oral phosphate binder administration, and early management of hyperkalemia). An additional 9 pts died on study, 8 pts from leukemia progression and 1 pt from intracranial bleed due to disease-related thrombocytopenia. Pharmacodynamics: Pre-treatment, 4 and 24 hrs post end-of-infusion samples of circulating leukemic blasts were obtained from 1 AML and 3 ALL pts. By Western blot, post-treatment decrease in Mcl-1 and increase in PARP cleavage were seen in all 4 pts at 4 hrs post-treatment, confirming that in vivo inhibition of CDKs was achieved, but recovery of Mcl-1 at 24 hrs was observed in all 4 pts, suggesting that inhibition was lost at 24 hrs. Decline in p-Rb was observed in 1 pt, while 2 pts had almost undetectable p-Rb levels at baseline. Conclusion: Dinaciclib showed anti-leukemia activity in this heavily pre-treated patient population. TLS was a notable toxicity, but was manageable in most pts with aggressive prophylaxis, monitoring and treatment. Early blast recovery and short duration of nadir observed on this study, combined with PK data showing a short t1/2 (1.5-3.3 hours) for dinaciclib and PD data demonstrating rapid reexpression of Mcl-1, support either use of longer infusion schedules (currently explored in solid tumors) or more frequent drug administration. Further exploration of dinaciclib dose and schedules in AML and ALL is planned. Disclosures: Gojo: Merck & Co.: Research Funding. Off Label Use: SCH 727965 (dinaciclib) is an investigational drug. Padmanabhan:Schering-Plough: Consultancy; Merck & Co.: Research Funding. Small:Merck & Co.: Employment, Equity Ownership. Zhang:Merck & Co.: Employment. Sadowska:Merck & Co.: Research Funding. Bannerji:Merck & Co.: Employment, Equity Ownership.

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Cold Agglutinin Disease Transfusion Practices in the United States: An Electronic Medical Record-Based Analysis

Blood ◽

10.1182/blood-2019-122209 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3690-3690

Author(s):

Jun Su ◽

Rajeshwari Punekar ◽

Jaime Morales Arias ◽

Nisha Jain

Keyword(s):

Medical Record ◽

Hemolytic Anemia ◽

Autoimmune Hemolytic Anemia ◽

Index Date ◽

Cold Agglutinin ◽

Cold Agglutinin Disease ◽

Employment Equity ◽

Medical Activity ◽

Equity Ownership ◽

Rbc Transfusion

Introduction Cold agglutinin disease (CAD) is a rare autoimmune hemolytic anemia (AIHA) accounting for 20% of all cases, with no approved therapies and limited management options for patients. CAD is characterized by immunoglobulin M-mediated erythrocyte agglutination, which triggers activation of the classical complement pathway leading to hemolysis and subsequent anemia. Red blood cell (RBC) transfusions are used as a supportive treatment in CAD to temporarily alleviate anemia, although the transfusion practices are variable among providers treating patients with CAD. Recent RBC transfusion guidelines from the AABB (formerly the American Association of Blood Banks) recommend that transfusions be administered with a restrictive threshold in most clinical scenarios (ie, transfusion is not indicated until hemoglobin [Hb] reaches 7-8 g/dL and/or patients exhibit anemia-related symptoms) to avoid associated complications such as acute reactions, alloantibody development, and hemochromatosis (Carson et al, JAMA, 2016; Carson et al, N Engl J Med, 2017). Because of the dearth of information available regarding trends in RBC transfusion practices among US hematologists, the objective of this longitudinal, retrospective, observational assessment of an electronic medical record database was to evaluate transfusion practices applied to patients with CAD in the US. Methods Patients were retrospectively identified from Optum® de-identified Electronic Health Record (EHR) dataset. Adult patients with ≥1 AIHA-related medical encounter between January 2007 and September 2018 (study period) and ≥3 mentions of CAD-related terms from physician notes ("cold agglutinin disease," "cold autoimmune hemolytic anemia," or "cold agglutinin hemoglobinuria") were included (Broome et al, Blood, 2017). The index date for each patient was the date of first mention of CAD during the study period. The baseline period was defined as the interval from the start of medical activity in the EHR database or study period (whichever occurred later) to the index date, and the follow-up period was defined as the interval from the index date to the end of the study period, end of medical activity, or death (whichever occurred earlier). The study sample was categorized into 2 study groups, the transfusion group (patients with CAD with ≥1 RBC transfusion after the index date) and the non-transfusion group (patients with CAD without any transfusions during the study period). Patients were further grouped based on the following Hb levels (g/dL): <8, ≥8 to ≤10, and >10 to ≤12. The closest Hb level prior to the most recent transfusion (within the prior 15 days and the lowest level) was used for the transfusion group and the lowest Hb level during the study period was used for the non-transfusion group. Descriptive statistics included mean, standard deviation, and median values for continuous variables and frequency (n and percent) for categorical variables. No adjustment was made for this descriptive analysis. Results A total of 903 patients with CAD were identified from the Optum EHR database; most patients were white (n=760 [84%]) and female (n=560 [62%]). Baseline demographics and clinical characteristics of each group can be found in the Table. Of the patients with CAD, 548 (61%) did not receive transfusions and 355 (39%) received ≥1 RBC transfusion. Among patients with CAD who received transfusions, 84% (n=297) had ≥2 RBC transfusions. Out of the 903 patients with CAD, 864 had Hb levels reported and 752 had Hb levels ≤12 g/dL. Forty-four percent (n=329/752) of those CAD patients received ≥1 RBC transfusion. When separated by Hb levels, 18% of patients with Hb >10 to ≤12 g/dL (n=19/108); 41% (n=88/216) of patients with Hb ≥8 to ≤10 g/dL; and 52% (n=222/428) of patients with Hb <8 g/dL received ≥1 RBC transfusion. Of the 423 (56%) patients with CAD and Hb levels ≤12 g/dL who did not receive RBC transfusions, 21% (n=89/423) had Hb levels >10 to ≤12 g/dL; 30% (n=128/423) had Hb levels ≥8 to ≤10 g/dL; and 49% (n=206/423) had Hb levels <8 g/dL. Conclusions Overall, patients with CAD are not a heavily transfused population. Even in those with a significantly decreased Hb (<8 g/dL), approximately half of them (49%) did not receive RBC transfusions. This suggests that the use of transfusions in patients with CAD may not reflect disease severity. Further prospective studies are needed to fully understand the impact of transfusions on patients with CAD. Disclosures Su: Sanofi Genzyme: Employment, Equity Ownership. Punekar:Sanofi: Employment, Equity Ownership. Morales Arias:Sanofi: Employment, Equity Ownership. Jain:Sanofi Genzyme: Employment, Equity Ownership.

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Mortality Among Patients with Cold Agglutinin Disease in the United States: An Electronic Health Record (EHR)-Based Analysis

Blood ◽

10.1182/blood-2019-122140 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4790-4790

Author(s):

Quentin A. Hill ◽

Rajeshwari Punekar ◽

Jaime Morales Arias ◽

Catherine M Broome ◽

Jun Su

Keyword(s):

Mortality Rate ◽

Research Funding ◽

Index Date ◽

Cold Agglutinin ◽

Control Cohort ◽

Cold Agglutinin Disease ◽

Employment Equity ◽

Equity Ownership ◽

And Control

Introduction Cold agglutinin disease (CAD) is a rare form of autoimmune hemolytic anemia (AIHA). It is characterized by both IgM-mediated agglutination of erythrocytes and hemolysis mediated by activation of the classical complement pathway. Recent studies have shown an increased risk of thromboembolic events (TE) in CAD patients. In addition, a recent analysis using the Danish National Patient Registry demonstrated a significant increase in mortality for CAD patients compared with matched individuals from the general population in Denmark (Bylsma et al, HemaSphere, 2018). Mortality among CAD patients has not been assessed in a US population. This study evaluated mortality risk in the largest cohort of CAD patients in the US. Methods Patients were retrospectively identified from the Optum® de-identified EHR dataset. Between January 2007 and September 2018 (study period), patients with ≥1 AIHA-related medical encounter and ≥3 mentions from physician notes of CAD-related terms ("cold agglutinin disease," "cold autoimmune hemolytic anemia," or "cold agglutinin hemoglobinuria") were included in the CAD cohort ("case"). For this cohort, the first mention of CAD terms was set as the index date. Patients without an AIHA-related medical encounter were included in the non-CAD cohort ("control"). For the control cohort, the index date was assigned based on the average occurrence of index date in the CAD population for the duration in the EHR database. For both cohorts, the baseline period was defined as the interval from the start of the medical activity in the EHR database or study period (whichever occurs later) to the index date, and the follow-up period was defined as the interval from the index date to the end of the study period, the end of medical activity, or death (whichever occurs earlier). The case and control cohorts were matched by age, gender, race, region, index year, and follow-up period using 1:5 nearest neighbor matching. Both cohorts were stratified according to the presence or absence of ≥1 TE during the study period. Mortality rate per 100,000 patients was calculated as the number of patients who died in each cohort divided by the number of patients in each cohort, from 2007 to 2018, multiplied by 100,000. Mortality rate was compared between matched cohorts using a Poisson test. An independent t-test was used to compare age at death between matched CAD and control groups; and time to death (starting from the index date) was analyzed using Kaplan-Meier curves and compared between matched cohorts using log-rank P test. Results In total, 651 CAD patients and 3,255 matched non-CAD controls were identified. Of these, 35% (n=228) of CAD patients and 20% (n=641) of non-CAD patients experienced ≥1 TE (P<0.001). Median age at index date for both cohorts was 72 years. Most patients were female (CAD 64%; non-CAD 65%) and Caucasian (CAD 85%; non-CAD 85%). Median follow-up duration was 42 months for the CAD cohort and 51 months for the control cohort. Mean (standard deviation [SD]) Elixhauser Comorbidity Index Score was 8.0 (4.9) for CAD patients and 4.5 (4.1) for matched controls. The overall mortality rate was significantly higher for the CAD cohort than the matched-control cohort (CAD: 17,512 vs non-CAD: 11,306; P<0.001). For patients that experienced ≥1 TE during the study period, the mortality rate in the CAD cohort was 23,684 compared with 15,913 in the matched-control cohort (P<0.001; Table 1). During the study period, 114 CAD patients and 368 matched non-CAD patients died. The mean (SD) age at death in the CAD cohort (77 [12] years) was lower compared with the matched controls (82 [8] years; P<0.001). For patients with ≥1 TE, mean (SD) age at death was 77 (13) years vs 82 (7) years for the CAD and control cohorts, respectively (P<0.001; Table 1). A Kaplan-Meier analysis demonstrated a significantly decreased survival probability among CAD patients compared with matched controls (P<0.001; Figure 1). In addition, CAD patients with ≥1 TE also had a significant decrease in survival when compared with matched controls with ≥1 TE (P<0.001). Conclusions CAD patients in the US have an increased mortality risk compared with a matched non-CAD population. The associated increased TE risk observed among CAD patients may be a contributing factor to this mortality. Further studies are needed to better define this association and elucidate other potential contributors to mortality in these patients. Disclosures Hill: Apellis: Honoraria; Novartis: Speakers Bureau; Bioverativ, a Sanofi company: Honoraria; Alexion: Research Funding. Punekar:Sanofi: Employment, Equity Ownership. Morales Arias:Sanofi: Employment, Equity Ownership. Broome:Cellphire: Research Funding; Alexion: Honoraria, Research Funding; Sanofi Genzyme: Honoraria, Research Funding; Incyte: Research Funding; Rigel: Research Funding. Su:Sanofi Genzyme: Employment, Equity Ownership.

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DNA Sequencing-Based Monitoring Of Serum Predicts Clinical Relapse Before CT Imaging in Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood.v122.21.1767.1767 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1767-1767 ◽

Cited By ~ 2

Author(s):

Mark Roschewski ◽

Stefania Pittaluga ◽

Kieron Dunleavy ◽

Katherine Kong ◽

Margaret Shovlin ◽

...

Keyword(s):

Lymph Node ◽

Dna Sequencing ◽

B Cell ◽

B Cell Lymphoma ◽

Circulating Tumor Dna ◽

Clinical Relapse ◽

Serum Samples ◽

Employment Equity ◽

Equity Ownership ◽

Pre Treatment

Abstract Background Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma with >40,000 cases annually in the US. While most patients are cured by frontline immunochemotherapy, up to 20% recur and salvage therapy is poor. Relapse detection relies on CT imaging, which is hampered by radiation exposure, high costs, high false positive rates, and infrequently detects relapse prior to symptoms. Novel methods to detect early relapse may improve outcomes. The LymphoSIGHT™ platform is a high-throughput DNA sequencing method that can detect minimal residual disease (MRD) of lymphoid malignancies in peripheral blood (PB) (Faham et al. Blood 2012) with a sensitivity of one lymphoma cell per million leukocytes. We showed it can also detect circulating tumor DNA in the PB of DLBCL pts (Armand Br J Haematol 2013). We assessed the sensitivity and specificity of LymphoSIGHT™ to detect circulating tumor DNA in serum after frontline therapy in DLBCL. Methods All pts were newly diagnosed DLBCL undergoing frontline EPOCH-based chemotherapy. Post-therapy surveillance included clinic visits, PB samples, and contrast CT scans at 3, 4, 6, 12 and 12 mos intervals for the first 5 years, respectively. Pre-treatment lymph node biopsies and PB samples collected at initial diagnosis and during surveillance visits were sent for VDJ amplification and MRD sequencing assessment. Using universal primer sets, we amplified immunoglobulin heavy and kappa chain (IGH and IGK) variable, diversity, and joining gene segments from genomic DNA in tumor biopsy and PB samples. Amplified products were sequenced, and tumor-specific clonotypes were identified for each pt based on their high frequency within the B-cell repertoire in the lymph node. The presence of the tumor-specific clonotype was then quantitated in PB samples. Results Twenty-seven pts, treated between July 1996 and July 2005, had adequate DNA from pre-treatment FFPE lymph node biopsies, and 25 (92.6%) had a high-frequency clonal rearrangement (MRD) detected. The median pt age was 49 years (20-75) and an IPI risk groups included 8 low, 3 low-intermediate, 11 high-intermediate and 3 high. The pts had a median of 12 surveillance CT scans (range 1-13) and 221 serum samples analyzed for MRD. Two pts progressed early after cycles 1 and 6. The first pt had progressive disease by CT scan after cycle 1 and remained MRD positive. The second pt remained MRD positive at the end of cycle 6, at which time a CT showed CR-unconfirmed, and progressed one month later. In the remaining 23 pts who achieved CR, seven relapsed at a median of 20.3 (range 5.6-151.6) months. The 16 pts in sustained CR had a median follow-up of 99.7 months (range 3.0-136.3) and none were MRD positive post-treatment (specificity 100%; 88.9-100%). There were 0 false positives in 200 post-treatment samples tested (specificity 100%, 99-100%). Of 7 relapsing pts, 6 had serum samples within 1 year of recurrence. Of these, 5 pts had a positive MRD (range 3.2 to 11.2 months) (sensitivity 83%; 44-98%) at a median of 9.2 months (range 3.2 to 11.2 months) prior to a positive CT scan (Figure). One relapsing pt only had a serum sample obtained 18 mos before CT recurrence and it was MRD negative. Conclusions Our data suggest that a DNA sequencing-based MRD test of serum can predict clinical relapse with high sensitivity and specificity in DLBCL prior to CT progression. MRD assay detection may significantly reduce the cost, inconvenience and toxicity of surveillance. Importantly, early detection of recurrence may significantly improve pt outcomes. Additional pts are being analyzed and updated results will be available. This work was supported by the Intramural Research Program of NCI at NIH and Sequenta, Inc. Disclosures: Kong: Sequenta: Employment, Equity Ownership. Zheng:Sequenta: Employment, Equity Ownership. Willis:Sequenta: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Faham:Sequenta: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

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TNT009, a Classical Complement Pathway Specific Inhibitor, Prevents Complement Dependent Hemolysis Induced By Cold Agglutinin Disease Patient Autoantibodies

Blood ◽

10.1182/blood.v122.21.42.42 ◽

2013 ◽

Vol 122 (21) ◽

pp. 42-42 ◽

Cited By ~ 4

Author(s):

Sandip Panicker ◽

Ju Shi ◽

Eileen Rose ◽

Sami Hussain ◽

Susan Tom ◽

...

Keyword(s):

Cold Agglutinin ◽

Dependent Manner ◽

Employment Equity ◽

Concentration Dependent Manner ◽

Classical Complement Pathway ◽

Equity Ownership ◽

Classical Complement ◽

Agglutinin Titer

Abstract Cold agglutinin disease (CAD) is an autoimmune hemolytic anemia in which autoantibodies bind to red blood cells (RBC) at temperatures below 37°C, resulting in activation of the classical complement pathway (CCP). CCP activation leads to hemolysis either intravascularly, by formation of the membrane attack complex, or extravascularly, when C3/C4 fragment deposition onto the RBC surface results in sequestration by the reticuloendothelial system. Here we describe the in vitro and in vivo activity of TNT003 and TNT009, inhibitors of a serine protease specific to the CCP, in pre-clinical models of CAD. TNT003 is a mouse monoclonal IgG2a antibody with sub-nanomolar affinity. TNT009 is the humanized form (IgG4) of TNT003 and retains affinity and specificity to its target. In vitro assays using IgM-sensitized sheep RBC and human or non-human primate (NHP) serum showed that TNT003 and TNT009 potently inhibited antibody-mediated hemolysis in a concentration dependent manner. Additionally, TNT003 and TNT009 inhibited CCP-mediated production of the anaphylatoxins C4a, C3a, and C5a. Flow cytometry analysis showed that both antibodies also prevented C3 fragment deposition on the RBC surface. Activity was proportional to the amount of serum used, and at 80% human or NHP serum, TNT003 completely inhibited hemolysis with an IC50 of ∼13 µg/mL. Using an ELISA-based assay, TNT003 inhibited C5b-9 deposition driven by the CCP but not by the alternative (CAP) or lectin (CLP) pathways. These data suggest that TNT003 and TNT009 are specific and potent inhibitors of the CCP. To demonstrate the utility of a CCP inhibitor in disease, we tested the ability of TNT003 and TNT009 to inhibit the CCP in ex vivo hemolysis assays using CAD patient autoantibodies. Type O- RBC were incubated in the presence of CAD plasma to sensitize the cells with autoantibody. RBC were then washed and 25% normal human serum (NHS) added as a source of complement. Thirteen of the seventeen CAD samples tested (76%) mediated C3 fragment deposition on the RBC surface as determined by flow cytometry. TNT003 significantly inhibited C3 fragment deposition by all patient samples that deposited complement (88 ± 2.6% inhibition, n = 13) with an average IC50 of 4.7 ± 0.4 µg/mL. One patient sample induced complement-dependent hemolysis of ∼50% of the RBC upon addition of NHS. In a concentration dependent manner, TNT003 and TNT009, but not control IgG, completely inhibited CAD autoantibody-mediated hemolysis (Fig. 1), as well as C4a, C3a and C5a generation. We further characterized each patient sample to determine cold agglutinin titer. We found that cold agglutinin titer correlated with the percent RBC staining positive for cell surface C3 fragments (R2 = 0.3566; p < .01; n = 17 samples; Fig. 2).Figure 1TNT003 and TNT009 inhibit CAD autoantibody-mediated hemolysisFigure 1. TNT003 and TNT009 inhibit CAD autoantibody-mediated hemolysisFigure 2Cold agglutinin titers correlate with C3 fragment deposition on RBCFigure 2. Cold agglutinin titers correlate with C3 fragment deposition on RBC Extravascular hemolysis of C3 fragment-coated RBC by liver macrophages is believed to be the primary mechanism of RBC destruction in CAD. We therefore tested the hypothesis that CAD patient plasma-induced C3 fragment deposition on RBC would promote phagocytosis by the monocytic cell line THP-1. We found that RBC sensitized in CAD plasma and exposed to NHS were engulfed in an FcgR-independent mechanism by THP-1 cells. RBC phagocytosis was significantly inhibited if NHS exposure occurred in the presence of TNT003 (100 µg/mL), but not a control IgG. The selective CCP inhibitory activity of TNT003 was evaluated in vivo in cynomolgus monkeys. TNT003 administered as a single IV injection at 30 mg/kg resulted in a Cmax of ∼330 µg/mL and detectable serum TNT003 thru ≥72 hours. Using an ELISA-based assay, we observed specific inhibition (≥95%) of the CCP for ≥72 hours. In contrast, CAP activity was modestly and transiently inhibited for 4 - 8 hours. At Cmax, endogenous C4a levels were reduced by >90% and returned to baseline levels by ≥96 hours. Serum samples containing TNT003 showed complete (100%) inhibition of hemolysis and C3 fragment deposition in vitro. CCP activity was completely restored to baseline after TNT003 concentrations fell below a predictable, threshold level. Collectively, these data indicate that TNT003 and TNT009 are potent and specific inhibitors of CCP activity and C3 fragment deposition in vitro and in vivo. These findings support the preclinical development of TNT009 for the treatment of CCP-mediated diseases including CAD. Disclosures: Panicker: True North Therapeutics, Inc.: Employment, Equity Ownership. Shi:True North Therapeutics, Inc.: Employment, Equity Ownership. Rose:True North Therapeutics, Inc.: Employment, Equity Ownership. Hussain:True North Therapeutics, Inc.: Employment, Equity Ownership. Tom:True North Therapeutics, Inc.: Employment, Equity Ownership. Strober:True North Therapeutics, Inc.: Employment. Sloan:True North Therapeutics, Inc.: Consultancy. Parry:True North Therapeutics, Inc.: Employment, Equity Ownership. Stagliano:True North Therapeutics, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

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ANX005, an Inhibitory Antibody Against C1q, Blocks Complement Activation Triggered By Cold Agglutinins in Human Disease

Blood ◽

10.1182/blood.v128.22.1265.1265 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1265-1265 ◽

Cited By ~ 10

Author(s):

Morie A. Gertz ◽

Haiyan Qiu ◽

Laura Kendall ◽

Mario Saltarelli ◽

Ted Yednock ◽

...

Keyword(s):

Complement Activation ◽

Research Funding ◽

Cold Agglutinin ◽

Employment Equity ◽

Cold Agglutinins ◽

Equity Ownership ◽

The Impact ◽

Human Rbcs ◽

Classical Complement

Abstract Cold agglutinin disease (CAD) is an autoimmune hemolytic anemia characterized by the presence of autoantibodies (cold agglutinins) that bind to red blood cells (RBC) at low temperatures. Cold agglutinin binding to RBCs results in antibody-mediated classical complement activation with deposition of complement C4 and C3 fragments onto the RBC surface. Complement-opsonized RBCs are removed from the circulation by macrophage-driven phagocytosis in the liver or spleen and via intravascular hemolysis following assembly of the terminal lytic complex (C5b - C9), together resulting in clinical anemia. C1q is the initiating molecule for cold agglutinin-mediated complement activation on the surface of human RBCs. We hypothesized that directly blocking C1q recruitment onto cold agglutinin-sensitized RBCs will prevent complement activation and opsonization and reduce hemolysis. To this end, we have developed a humanized monoclonal antibody (ANX005) that binds with high-affinity (~10 pM) to C1q and blocks classical complement activation & hemolysis in an in vitro sheep RBC assay. We evaluated the impact of ANX005 on hemolysis and complement deposition on human RBCs that were pre-sensitized with sera from CAD subjects. ANX005 showed a dose-dependent reduction in hemolysis using both individual and pooled CAD sera as the source of cold agglutinin. We further demonstrated that C1q blockade led to a robust reduction in C4 and C3 fragment deposition onto human RBCs. These results demonstrate that C1q inhibition is an effective way to impede C4 and C3 activation and downstream assembly of the lytic complex in sera from CAD patients, and support the clinical development of ANX005 in CAD and other antibody-mediated diseases. Disclosures Gertz: Prothena Therapeutics: Research Funding; Sandoz Inc: Honoraria; NCI Frederick: Honoraria; Celgene: Honoraria; Med Learning Group: Honoraria, Speakers Bureau; Research to Practice: Honoraria, Speakers Bureau; Alnylam Pharmaceuticals: Research Funding; Novartis: Research Funding; Ionis: Research Funding; Annexon Biosciences: Research Funding; GSK: Honoraria. Qiu:Annexon Biosciences: Employment, Equity Ownership. Kendall:Annexon Biosciences: Employment, Equity Ownership. Saltarelli:Mallinckrodt: Equity Ownership; Abbvie: Equity Ownership; Annexon Biosciences: Employment, Equity Ownership, Patents & Royalties. Yednock:Annexon, Inc: Employment, Equity Ownership. Sankaranarayanan:Annexon Biosciences: Employment, Equity Ownership.

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Phenotype Does Not Always Equal Function: HDAC Inhibitors and UM171, but Not SR1, Lead to Rapid Upregulation of CD90 on Non-Engrafting CD34+CD90-Negative Human Cells

Blood ◽

10.1182/blood.v130.suppl_1.659.659 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 659-659

Author(s):

Kevin A. Goncalves ◽

Megan D. Hoban ◽

Jennifer L. Proctor ◽

Hillary L. Adams ◽

Sharon L. Hyzy ◽

...

Keyword(s):

In Vitro Culture ◽

Small Molecule ◽

Hdac Inhibitors ◽

Employment Equity ◽

Nsg Mice ◽

Cd34 Cells ◽

Equity Ownership ◽

Human Hscs

Abstract Background. The ability to expand human hematopoietic stem cells (HSCs) has the potential to improve outcomes in HSC transplantation and increase the dose of gene-modified HSCs. While many approaches have been reported to expand HSCs, a direct comparison of the various methods to expand transplantable HSCs has not been published and clinical outcome data for the various methods is incomplete. In the present study, we compared several small molecule approaches reported to expand human HSCs including HDAC inhibitors, the aryl hydrocarbon antagonist, SR1, and UM171, a small molecule with unknown mechanism, for the ability to expand phenotypic HSC during in vitro culture and to expand cells that engraft NSG mice. Although all strategies increased the number of phenotypic HSC (CD34+CD90+CD45RA-) in vitro, SR1 was the most effective method to increase the number of NOD-SCID engrafting cells. Importantly, we found that HDAC inhibitors and UM171 upregulated phenotypic stem cell markers on downstream progenitors, suggesting that these compounds do not expand true HSCs. Methods. Small-molecules, SR1, HDAC inhibitors (BG45, CAY10398, CAY10433, CAY10603, Entinostat, HC Toxin, LMK235, PCI-34051, Pyroxamide, Romidepsin, SAHA, Scriptaid, TMP269, Trichostatin A, or Valproic Acid) and UM171 were titrated and then evaluated at their optimal concentrations in the presence of cytokines (TPO, SCF, FLT3L, and IL6) for the ability to expand human mobilized peripheral blood (mPB)-derived CD34+ cells ex vivo . Immunophenotype and cell numbers were assessed by flow cytometry following a 7-day expansion assay in 10-point dose-response (10 µM to 0.5 nM). HSC function was evaluated by enumeration of colony forming units in methylcellulose and a subset of the compounds were evaluated by transplanting expanded cells into sub-lethally irradiated NSG mice to assess engraftment potential in vivo . All cells expanded with compounds were compared to uncultured or vehicle-cultured cells. Results. Following 7 days of expansion, SR1 (5-fold), UM171 (4-fold), or HDAC inhibitors (>3-35-fold) resulted in an increase in CD34+CD90+CD45RA- number relative to cells cultured with cytokines alone; however, only SR1 (18-fold) and UM171 (8-fold) demonstrated enhanced engraftment in NSG mice. Interestingly, while HDAC inhibitors and UM171 gave the most robust increase in the number and frequency of CD34+CD90+CD45RA- cells during in vitro culture, these methods were inferior to SR1 at increasing NSG engrafting cells. The increase in CD34+CD90+CD45RA- cells observed during in vitro culture suggested that these compounds may be generating a false phenotype by upregulating CD90 and down-regulating CD45RA on progenitors that were originally CD34+CD90-CD45RA+. We tested this hypothesis by sorting CD34+CD90-CD45RA+ cells and culturing these with the various compounds. These experiments confirmed that both HDAC inhibitors (33-100 fold) and UM171 (28-fold) led to upregulation of CD90 on CD34+CD90-CD45RA+ cells after 4 days in culture. Since approximately 90% of the starting CD34+ cells were CD90-, these data suggest that most of the CD34+CD90+CD45RA- cells in cultures with HDAC inhibitors and UM171 arise from upregulation of CD90 rather than expansion of true CD34+CD90+CD45RA- cells and may explain the disconnect between in vitro HSC phenotype and NSG engraftment in vivo . This was further confirmed by evaluation of colony forming unit frequency of CD34+CD90-CD45RA+ cells after culture with compounds. Conclusions. We have showed that AHR antagonism is optimal for expanding functional human HSCs using the NSG engraftment model. We also demonstrated that UM171 and HDAC inhibitors upregulate phenotypic HSC markers on downstream progenitors. This could explain the discrepancy between impressive in vitro phenotypic expansion and insufficient functional activity in the NSG mouse model. Therefore, these data suggest caution when interpreting in vitro expansion phenotypes without confirmatory functional transplantation data, especially as these approaches move into clinical trials in patients. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership. Hoban: Magenta Therapeutics: Employment, Equity Ownership. Proctor: Magenta Therapeutics: Employment, Equity Ownership. Adams: Magenta Therapeutics: Employment, Equity Ownership. Hyzy: Magenta Therapeutics: Employment, Equity Ownership. Boitano: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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Highly Differentiated Anti-CD47 Antibody, AO-176, Potently Inhibits Hematologic Malignancies Alone and in Combination

Blood ◽

10.1182/blood-2019-126298 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1844-1844

Author(s):

John Richards ◽

Myriam N Bouchlaka ◽

Robyn J Puro ◽

Ben J Capoccia ◽

Ronald R Hiebsch ◽

...

Keyword(s):

Multiple Myeloma ◽

Combination Therapy ◽

Tumor Growth ◽

Growth Inhibition ◽

Tumor Cells ◽

Tumor Growth Inhibition ◽

Employment Equity ◽

Equity Ownership

AO-176 is a highly differentiated, humanized anti-CD47 IgG2 antibody that is unique among agents in this class of checkpoint inhibitors. AO-176 works by blocking the "don't eat me" signal, the standard mechanism of anti-CD47 antibodies, but also by directly killing tumor cells. Importantly, AO-176 binds preferentially to tumor cells, compared to normal cells, and binds even more potently to tumors in their acidic microenvironment (low pH). Hematological neoplasms are the fourth most frequently diagnosed cancers in both men and women and account for approximately 10% of all cancers. Here we describe AO-176, a highly differentiated anti-CD47 antibody that potently targets hematologic cancers in vitro and in vivo. As a single agent, AO-176 not only promotes phagocytosis (15-45%, EC50 = 0.33-4.1 µg/ml) of hematologic tumor cell lines (acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and T cell leukemia) but also directly targets and kills tumor cells (18-46% Annexin V positivity, EC50 = 0.63-10 µg/ml) in a non-ADCC manner. In combination with agents targeting CD20 (rituximab) or CD38 (daratumumab), AO-176 mediates enhanced phagocytosis of lymphoma and multiple myeloma cell lines, respectively. In vivo, AO-176 mediates potent monotherapy tumor growth inhibition of hematologic tumors including Raji B cell lymphoma and RPMI-8226 multiple myeloma xenograft models in a dose-dependent manner. Concomitant with tumor growth inhibition, immune cell infiltrates were observed with elevated numbers of macrophage and dendritic cells, along with increased pro-inflammatory cytokine levels in AO-176 treated animals. When combined with bortezomib, AO-176 was able to elicit complete tumor regression (100% CR in 10/10 animals treated with either 10 or 25 mg/kg AO-176 + 1 mg/kg bortezomib) with no detectable tumor out to 100 days at study termination. Overall survival was also greatly improved following combination therapy compared to animals treated with bortezomib or AO-176 alone. These data show that AO-176 exhibits promising monotherapy and combination therapy activity, both in vitro and in vivo, against hematologic cancers. These findings also add to the previously reported anti-tumor efficacy exhibited by AO-176 in solid tumor xenografts representing ovarian, gastric and breast cancer. With AO-176's highly differentiated MOA and binding characteristics, it may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Disclosures Richards: Arch Oncology Inc.: Employment, Equity Ownership, Other: Salary. Bouchlaka:Arch Oncology Inc.: Consultancy, Equity Ownership. Puro:Arch Oncology Inc.: Employment, Equity Ownership. Capoccia:Arch Oncology Inc.: Employment, Equity Ownership. Hiebsch:Arch Oncology Inc.: Employment, Equity Ownership. Donio:Arch Oncology Inc.: Employment, Equity Ownership. Wilson:Arch Oncology Inc.: Employment, Equity Ownership. Chakraborty:Arch Oncology Inc.: Employment, Equity Ownership. Sung:Arch Oncology Inc.: Employment, Equity Ownership. Pereira:Arch Oncology Inc.: Employment, Equity Ownership.

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Differential Effects of Milatuzumab On Human Antigen-Presenting Cells in Comparison to Malignant B Cells.

Blood ◽

10.1182/blood.v114.22.2744.2744 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2744-2744

Author(s):

Xiaochuan Chen ◽

Rhona Stein ◽

Chien-Hsing Chang ◽

David M. Goldenberg

Keyword(s):

T Cell ◽

B Cells ◽

Cell Lines ◽

Hairy Cell ◽

Employment Equity ◽

Cross Presentation ◽

Equity Ownership ◽

Antigen Presenting

Abstract Abstract 2744 Poster Board II-720 Introduction: The humanized anti-CD74 monoclonal antibody (mAb), milatuzumab, is in clinical evaluation as a therapeutic mAb for non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and multiple myeloma after preclinical evidence of activity in these tumor types. In addition to its expression in malignant cells, CD74 is also expressed in normal B cells, monocytes, macrophages, Langerhans cells, follicular and blood dendritic cells. A question therefore arises whether milatuzumab is toxic to or affects the function of these immune cells. This has important implications, not only for safe therapeutic use of this mAb, but also for its potential application as a novel delivery modality for in-vivo targeted vaccination. Methods: We assessed the binding profiles and functional effects of milatuzumab on human antigen-presenting cell (APC) subsets. Studies on the effect of milatuzumab on antigen presentation and cross-presentation are included. In addition, binding and cytotoxicity on a panel of leukemia/lymphoma cell lines and CLL patient cells were tested to demonstrate the range of malignancies that can be treated with this mAb. Results: Milatuzumab bound efficiently to different subsets of blood dendritic cells, including BDCA-1+ myeloid DCs (MDC1), BDCA-2+ plasmacytoid DCs (PDC), BDCA-3+ myeloid DCs (MDC2), B lymphocytes, monocytes, and immature DCs derived from human monocytes in vitro, but not LPS-matured DCs, which correlated well with their CD74 expression levels. In the malignant B-cells tested, milatuzumab bound to the surface of 2/3 AML, 2/2 mantle cell (MCL), 4/4 ALL, 1/1 hairy cell leukemia, 2/2 CLL, 7/7 NHL, and 5/6 multiple myeloma cell lines, and cells of 4/6 CLL patient specimens. Significant cytotoxicity (P<0.05) was observed in 2/2 MCL, 2/2 CLL, 3/4 ALL, 1/1 hairy cell, 2/2 NHL, and 2/2 MM cell lines, and 3/4 CD74-positive CLL patient cells, but not in the AML cell lines following incubation with milatuzumab. In contrast, milatuzumab had minimal effects on the viability of DCs or B cells that normally express CD74. The DC maturation and DC-mediated T-cell functions were not altered by milatuzumab treatment, which include DC-induced T-cell proliferation, CD4+CD25+FoxP3+ Treg expansion, and CD4+ naïve T-cell polarization. Moreover, milatuzumab had little effect on CMV-specific CD8- and CD8+ T cell interferon-g responses of peripheral blood mononuclear cells stimulated in vitro with CMV pp65 peptides or protein, suggesting that milatuzumab does not influence antigen presentation or cross-presentation. Conclusion: These results demonstrate that milatuzumab is a highly specific therapeutic mAb against B-cell malignancies with potentially minimal side effects. It also suggests that milatuzumab may be a promising novel delivery mAb for in vivo targeted vaccinations, given its efficient binding, but lack of cytotoxicity and functional disruption on CD74-expressing normal APCs. (Supported in part by NIH grant PO1-CA103985.) Disclosures: Chang: Immunomedics Inc.: Employment, Equity Ownership, Patents & Royalties. Goldenberg:Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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