Serum Rituximab Research Articles

Use of E-64 cysteine protease inhibitor for the recombinant protein production in Tetrahymena thermophila

Tetrahymena thermophila is an alternative organism for recombinant protein production. However, the production efficiency in T. thermophila is quite low mainly due to the rich cysteine proteases. In this study, we studied whether supplementation of the E-64 inhibitor to T. thermophila cultures increases the recombinant protein production efficiency without any toxic side effects. Our study showed that supplementation of E-64 had no lethal effects on T. thermophila cells in flask culture at 30 °C and 38 °C. In vitro protease activity analysis using secretome as protease enzyme source from E-64-supplemented cell cultures showed a reduced protein substrate degradation using bovine serum albumin, rituximab, and milk lactoglobulin proteins. E-64 also prevented proteolysis of the recombinantly produced and secreted TtmCherry2-sfGFP fusion protein at some level. This reduced inhibitory effect of E-64 could be due to genetic compensation of the inhibited proteases. As a result, the 5 µM concentration of E-64 was found to be a non-toxic protease inhibitory supplement to improve extracellular recombinant protein production efficiency in T. thermophila. This study suggests that the use of E-64 may increase the efficiency of extracellular recombinant protein production by continuously reducing extracellular cysteine protease activity during cultivation.

Abstract 4990: Genotoxic chemotherapy impedes antibody-mediated complement dependent cytotoxicity (CDC), via a Chk1-CD59 axis

Abstract Monoclonal antibodies such as rituximab exert targeted tumor cell lysis via complement dependent cytotoxicity (CDC). These antibodies are often combined with chemotherapy in clinical treatment protocols. However, the impact of chemotherapy on cell-intrinsic pathways that regulate antibody-mediated CDC is not known. Through a phage display screen, we identified that genotoxic stress caused upregulation of a membrane complement regulatory protein (mCRP), CD46, prompting further investigation into its implications and mechanism. Using cell lines from diffuse large B cell lymphoma (DLBCL) as our model, we confirmed that genotoxic chemotherapy upregulates CD46, as well as other mCRPs such as CD55 and CD59. We hypothesized that upregulated mCRPs could impede Rituximab-mediated CDC. We assessed this in-vitro using rituximab and complement human serum. Aligned with our hypothesis, chemotherapy treatment of DLBCL cells significantly impeded rituximab-induced CDC. Using blocking antibodies to each mCRP, reversal of the effect of chemotherapy on CDC only occurred after co-treatment with a CD59 blocking antibody, suggesting that CD59 is a key player for chemotherapy-invoked resistance to CDC. To further elucidate the underlying cellular pathway, we performed a high-throughput small molecule screen using rituximab-induced CDC as a readout, with or without cotreatment of a genotoxic chemotherapeutic (etoposide). We identified Chk1 inhibitors as consistent top rescuers. qPCR and flow cytometry revealed that Chk1 inhibition prevented the upregulation of mCRPs transcriptionally. Sp1 is a known transcription factor driving constitutive CD59 expression. Inhibiting Sp1 similarly prevented chemotherapy-induced upregulation of CD59 and resistance to CDC. However, CHIP-qPCR showed no increased binding of total Sp1 protein to the CD59 promoter after chemotherapy. Instead, positive co-immunoprecipitation of pATM and p300 with Sp1 only with etoposide and not with co-treatment of Chk1 inhibitor suggests Chk1-mediated activation of Sp1. Mechanistically, we have found that chemotherapy-activated Chk1 modulates Sp1-mediated CD59 transcription and that the induced upregulation of CD59 is able to prevent antibody-mediated CDC. Overall, these results indicate novel immune regulation by DNA damage response, particularly in the context of complement regulatory proteins. Our results advocate consideration when using simultaneous administration of chemotherapy with CDC-inducing monoclonal antibodies, and suggest Chk1 blockade as a possible therapeutic addition. Citation Format: Allison S. Chan, Patrick W. Jaynes, Nurulhuda B. Mustafa, Akshaya A. Anbuselvan, Charmaine Z. Ong, Anand D. Jeyasekharan. Genotoxic chemotherapy impedes antibody-mediated complement dependent cytotoxicity (CDC), via a Chk1-CD59 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4990.

Low Dose Rituximab Efficiently Clears Circulating CLL Cells and Maintains Sensitivity to Antibody Dependent Cellular Phagocytosis (ADCP)

Introduction: We have recently shown that high frequency (2 x week) low dose (50 mg)(HFLD) rituximab (RTX) and acalabrutinib is highly effective initial therapy for patients (n=38) with progressive CLL (ClinicalTrials.gov NCT03788291, PMID:36689726). The first dose of 50 mg of IV rituximab administered at 25 mg/h decreased the median circulating CLL cell count by 84% from pre-treatment baseline at 1h with no further decrease in the count during the remainder of the infusion (PMID:37003030). During the first hour of infusion, median CLL cell membrane CD20 levels decreased to 65% of baseline and were 41% of baseline at the end of the infusion. Median serum rituximab concentration was 2 mcg/ml at 1h and 10 mcg/ml at the end of the infusion. Median complement (CH50) concentration decreased to 91% of baseline at 1 h and to 81% of baseline at the end of the infusion. The cessation of clearance of CLL cells from the circulation within 1h of starting rituximab therapy is thus unlikely to be caused by low rituximab levels, decreased CLL cell CD20 expression or low serum complement levels. We and others have previously shown that ADCP is the primary mechanism of clearance of anti-CD20 mAb opsonized B cells in both humans and mice. We hypothesized that clearance of circulating CLL cells decreases because residual circulating CLL cells are resistant to mAb induced ADCP. To test this hypothesis, we measured in vitro ADCP sensitivity of CLL cells sampled from patients before and after rituximab treatment. Methods: Blood specimens were collected from 13 patients prior to starting therapy (C1D1_pre), 1h after start of IV rituximab infusion (C1D1_1h), at end of rituximab infusion (C1D1_post), and 48 h after initiation of therapy (C1D3_pre). Sensitivity of circulating CLL cells to in vitro ADCP was measured using live cell time-lapse high-content microscopy imaging of phagocytosis of CLL cells by human monocyte derived macrophages (hMDM) as previously described (PMID:32005699). Phagocytosis was quantified using a void index and sensitivity of samples was compared using the area under the curve of the 2h void index plot (AUC). Experiments were done in triplicate with co-cultures of hMDM and CLL cells without mAb as controls. Rituximab (10 mcg/ml) was used to test CD20 mAb mediated ADCP and alemtuzumab (10 mcg/ml) to test non-CD20 mediated ADCP. Results: Figure (median and 95% confidence intervals) shows that neither rituximab nor alemtuzumab induced ex-vivo ADCP as measured by median void index AUC was significantly altered by prior in vivo exposure of CLL cells to rituximab (Wilcoxon Match-Paired Sign test; all p>0.05). Discussion: The rapid decrease in circulating CLL cells induced by IV rituximab occurred only within the first hour after initiation of mAb infusion. Our measurements show that serum rituximab and complement concentrations and CLL cell membrane CD20 levels should be adequate to support additional CLL cell clearance after 1h. We now demonstrate that residual circulating CLL cells remain sensitive to in vitro ADCP by both rituximab and alemtuzumab. This suggests that despite reduced levels of surface antigen, circulating cells remain sensitive to mAb mediated clearance. Alternative explanations for the limited clearance of circulating CLL cells include: 1. Saturation of the limited cytotoxic capacity of the innate immune effector cells (Kupffer cells and splenic macrophages) compatible with our previously reported data on finite macrophage ADCP capacity (PMID:32556153); 2. Mobilization of CLL cells from lymphoid tissue; and 3. Temporary sequestration of rituximab opsonized CLL cells in tissue (e.g. spleen or lungs), which is unlikely given the observed decrease in lymph node size following initiation of therapy. Ongoing research is testing these hypotheses.

#5710 CHARACTERIZATION OF RESPONSE TO TREATMENT WITH RITUXIMAB IN PATIENTS WITH PRIMARY GLOMERULOPATIES

Abstract Background and Aims Rituximab (RTX) is an anti-CD20 monoclonal antibody used in renal diseases with glomerular involvement, which usually present with proteinuria. The doses used in this pathologies are extrapolated from other diseases such as rheumatoid arthritis, but the optimal regimen in this population has not been well defined. Objective To describe the behaviuor of RITUXIMAB in patients with primary glomerulopathies in whom its use is indicated. Method Descriptive low-intervention single-center clinical trial approved by the Spanish Medicines Agency (NEFRTX, EUDRACT 2020-000484-23. Patients treated with rituximab were included according to medical criteria, prior approval by the Pharmacy Commission. The regimen used was 1g every 15 days, or only 1gr dose with premedication according to clinical practice guidelines. 24-hour blood and urine samples were collected on days 1 (post-rituximab), 7, 15 (pre and post-rituximab), 28, and 45. Serum rituximab was determined using ELISA (Lisa-Traker®-Rituximab, Theraradiag®). Clinical and pharmacokinetic data analyzes were performed considering a single compartment model by non-linear regression with Winnolin® to obtain clearance (Cl), half-life (t1/2), maximum concentration (Cmax) and volume of distribution (Vd). Some quantitative results are expressed as mean ± standard deviation. Linear regression was used to establish correlations. Results The results of 5 patients were analyzed, 2 women and 3 men, with a mean age of 68.8 ± 17.02 years of 72 years and a diagnosis of membranous GN (3), ANCA MPO GN (1), focal and segmental GN (1). Renal function CKD EPI: GFR between 45- 60 ml/min N = 2 GFR >60 ml/min N = 3. According to clinical practice guidelines, patients received 1g doses, 2/5 patients received a second dose on day 15. The evaluation parameters were: proteinuria, CD 19+ lymphocyte depletion, Antibodies titers and histological characteristics as appropriate. levels of rituximab in urine samples after the rituximab administration (15, 30, 180, 365 days after) were determinate. All the patients presented proteinuria, with a mean of 11±14.6 g in 24 h urine. Mean Rituximab clearance was 0.676±0.431 ml/h/kg, distribution volume was 222.35±140.52 mL/Kg and Cmax 69.66±26.14 mcg/mL and t1/2 269.66± 229.71 h (11 days approximately). There is a correlation between proteinuria and rituximab half-life (p = 0.077), as well as between proteinuria and clearance creatinine (p = 0.975) suggesting that half life of rituximab is lower if proteinuria increases (Fig. 1). All presented lymphocyte depletion of CD 19 at 45 days. However, those with more proteinuria >2.5 g (N = 3) in those 45 days, after 6 months of exposure to RTX, progressive repopulation of CD19 was observed, which persisted in the year of follow-up. Conclusion In patients with high proteinuria, rituximab has a t1/2 lower than the 21–28 days described in the data sheet, which may suggest that patients with nephrotic syndrome may require higher doses to ensure the effectiveness of treatment with rituximab. Repopulation of CD19 in 45 days could be a reliable indicator of non response in patients with nephrotic proteinuria.

Rituximab induced cytokine release with high serum IP-10 (CXCL10) concentrations is associated with infusion reactions

Monoclonal antibody induced infusion reactions (IRs) can be serious and even fatal. We used clinical data and blood samples from 37 treatment naïve patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) initiating therapy for progressive disease with a single 50 mg dose of intravenous (IV) rituximab at 25 mg/h. Twenty-four (65 %) patients had IRs at a median of 78 min (range 35–128) and rituximab dose of 32 mg (range 15–50). IR risk did not correlate with patient or CLL characteristics, CLL counts or CD20 levels, or serum rituximab or complement concentrations. Thirty-five (95 %) patients had cytokine release response with a ≥ 4-fold increase in serum concentration of ≥ 1 inflammatory cytokine. IRs were associated with significantly higher post-infusion serum concentrations of gamma interferon induced cytokines IP-10, IL-6 and IL-8. IP-10 concentrations increased ≥ 4-fold in all patients with an IR and were above the upper limit of detection (40,000 pg/ml) in 17 (71 %). In contrast, to only three (23 %) patients without an IR had an ≥ 4-fold increase in serum concentrations of IP-10 (highest 22,013 pg/ml). Our data suggest that cytokine release could be initiated by activation of effector cells responsible for clearance of circulating CLL cells with IRs occurring in those with higher levels of gamma interferon induced cytokines. These novel insights could inform future research to better understand and manage IRs and understand the role of cytokines in the control of cytotoxic immune responses to mAb.

Rituximab Induces an Inflammatory Cytokine Response with Increased IP-10 (CXCR10) in Patients with Infusion Reactions

Most patients initiating anti-CD20 monoclonal antibody (mAb) therapy for CLL have a disruptive infusion reaction that can cause serious complications and be fatal. A better understanding of the risk factors and etiology of infusion reactions is required to improve preventative measures and management. We report a study to test the hypothesis that IV rituximab induces an inflammatory cytokines response with differences in individual cytokine concentrations correlating with infusion reactions. We designed a clinical trial (NCT03788291) for initial treatment of patients with progressive CLL that provided clinical data and blood specimens from 37 patients treated with 50 mg of IV rituximab infused at 25 mg/h. Blood specimens were collected immediately prior to initiation of rituximab (baseline), one hour later (1h), at the end of the infusion (post-infusion) and at 48 h after initiation of therapy (48h). Infusion reactions (CTCAE grade ≥2 events) were managed by interrupting the infusion and initiating symptomatic treatment. We measured circulating CLL cell counts and CD20 levels, and serum rituximab concentration, complement levels, and cytokine concentrations of IFN-γ, IL-10, IL-18, IL-2, IL-4, IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, and TNF-α using standard methods. Twenty-four (65%) patients had 25 infusion reactions (all CTCAE grade 2) at a median of 78 minutes (range 35-128) after initiation of rituximab therapy (median dose 32 mg, range 15-50). All patients completed the 50 mg rituximab infusion (median time 150 minutes, range 119-195). Patient and CLL characteristics including baseline CLL cell counts did not predict the risk of infusion reactions. Median CLL counts at 1h decreased to 14.7% of baseline with no further changes post-infusion and no significant difference between patients with or without infusion reactions (60x109/L vs. 45x109/L, p=0.61). Median CLL cell membrane CD20 levels (x103 molecules/cell) decreased from 4.77 at baseline to 3.10 at 1h (p=0.002) and 1.59 post-infusion (p<0.0001 vs. baseline and 1h) with no significant differences between patients with or without an infusion reaction. The median estimated number of circulating CLL cell membrane CD20 molecules (x1012 molecules/L) decreased from 325.8 at baseline to 34.1 at 1h (p<0.0001) and 14.5 post-infusion (p<0.0001 vs. baseline, p<0.03 vs. 1h) with no significant differences between patients with or without an infusion reaction. There was no correlation between risk of infusion reaction and serum levels of rituximab or serum complement. Thirty-five (95%) patients had a ≥4 fold increase in serum concentration of ≥1 cytokine with resolution of the cytokine response at 48h. All patients with an infusion reaction had significantly higher median post-infusion serum concentrations of IP-10, IL-6 and IL-8. All 17 patients with IP-10 serum concentrations above the limit of detection (40,000 pg/ml) post-infusion had an infusion reaction, and all 24 patients with an infusion reaction had a ≥4 fold increase in serum IP-10 concentrations. Of 13 patients without an infusion reaction, the highest measured serum IP-10 concentration was 22,013 pg/ml and only 3 (23%) had ≥4 fold increases in serum IP-10 concentrations. Slow infusion of a reduced dose of rituximab induced a cytokine response in 95% of patients and therapy disrupting infusion reactions occurred at a comparable rate (65%) to standard rituximab therapy. Infusion reactions were associated with increased levels of IP-10, IL-6 and IL-8. IP-10 levels were increased by ≥4 fold in all patients with an infusion reaction compared to 23% without an infusion reaction. These data suggest that rituximab infusion reactions are caused by inflammatory cytokines produced by cytotoxic effector cells activated by rituximab independent of the pre-treatment amount of circulating CLL cells or CD20 levels, or efficacy of rituximab in reducing the circulating CLL cell count. Infusion reactions occurred after at least 35 minutes of therapy concurrent with a rapid decrease in circulating CLL cells. This observation suggests that cytokine release could be a result of activation of effector cells responsible for CLL cell elimination. Our data provides novel insights into the potential mechanism of infusion reactions and could form the basis of further research to determine methods to control this clinically important and therapy limiting complication.

Temperature-modulated antibody drug separation using thermoresponsive mixed polymer brush-modified stationary phase

Recently, antibody drugs have been investigated for medical treatments of intractable diseases. An effective antibody drug separation method is in demand for production of such drugs. We developed an antibody drug separation material using a mixed polymer brush composed of poly(N-isopropylacrylamide) (PNIPAAm), which is a thermoresponsive polymer, and poly(4-vinylpyridine) (P4VP), which is an antibody-affinity polymer, for temperature modulation and adsorption of the antibody drug rituximab that results in its separation. A mixed polymer brush was prepared on silica bead surfaces by radical polymerization to modify P4VP, followed by atom transfer radical polymerization to modify PNIPAAm. The prepared mixed polymer brush was characterized by CHN elemental analysis, chloride analysis, nitrogen adsorption, thermogravimetric analysis, differential thermal analysis, differential scanning calorimetry, Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, transmission electron microscopy, and zeta potential measurement. Temperature-modulated adsorption of rituximab on the mixed polymer brush was achieved by concealing and exposing P4VP through the extension and shrinkage of PNIPAAm in the brush, and was investigated using mixed polymer brush-modified silica beads as chromatographic packing materials. By utilizing the specific properties of mixed polymer brushes, rituximab and bovine serum albumin could be separated by simply changing the column temperature. Thus, the mixed polymer brush-modified beads prepared in this study may be useful for purifying rituximab by changing the temperature.

P243 Anti-rituximab antibodies demonstrate neutralising capacity, associate with lower circulating drug levels and early relapse in patients undergoing treatment for systemic lupus erythematosus

Abstract Background/Aims A major limitation of biologic therapy is formation of anti-drug antibodies (ADA). We previously demonstrated that ADA to rituximab are more prevalent in patients undergoing treatment for systemic lupus erythematosus (SLE) than rheumatoid arthritis and vasculitis, and predict subsequent infusion reactions. However, little is known regarding the long-term dynamics of ADA to rituximab. In this study we evaluated the longitudinal impact of ADA positivity in terms of drug kinetics, treatment response and neutralising capacity in SLE. Methods Patients with SLE undergoing treatment with rituximab were recruited to this study (n = 35). Serum samples were collected at the following intervals post-treatment; 1-3 months, 6 months, 12 months, 36 months (n = 114). Clinical and laboratory data was collected pre-treatment and at each time point. ADA were detected using an electrochemiluminescent immunoassay. Serum rituximab levels were measured via ELISA. A complement dependent cytotoxic assay was used to determine neutralising capability of ADA in a subgroup of positive samples (n = 38). Results ADA were found to be persistently positive over the 36-month follow-up period in 64.3% of patients. There was no significant difference in baseline disease activity (BILAG / SLEDAI-2K) between those who were subsequently ADA positive vs negative. ADA positive patients were younger at diagnosis compared with ADA negative (mean 22.50 ± 9.10 vs 37.29 ± 11.31 years, p = 0.002). Multivariate logistic regression found older age at diagnosis was associated with reduced incidence of persistent ADA with a 22% decrease in risk for each addition year after diagnosis (p = 0.03). ADA positive patients had a significantly lower C3 level at baseline (mean 0.61 ± 0.23 vs 0.87 ± 0.30, p = 0.026), which remained lower at each subsequent time point post-treatment up to 12 months. ADA titres peaked earlier and remained higher in those who had been treated with rituximab previously compared with those who were rituximab naive (median 132 AU/ml vs 7 AU/ml at 1-3 months). At 6 months post-treatment, patients with detectable ADA had a significantly lower circulating drug level than ADA negative (p = 0.0018). In terms of clinical response, ADA positive patients had an initial significant improvement in disease activity (SLEDAI-2K) by 3 months (p &amp;lt; 0.001). However, response was not maintained at 12 months. In comparison, ADA-negative patients showed a significant improvement in SLEDAI-2K at 6 months and this was maintained across the 36-month follow-up period. BILAG defined relapse within 12 months of treatment was only seen in ADA positive patients. Finally, all ADA positive samples were found to neutralise rituximab in vitro. Conclusion ADA to rituximab were common and persisted over the 36 month period of this study. ADA associated with earlier drug elimination, increased relapse rates and demonstrated neutralising capacity suggesting that ADA could be a significant limitation to sustained response to treatment. Disclosure C. Wincup: Grants/research support; Lupus UK, Versus Arthritis, British Society for Rheumatology. N. Dunn: None. C. Ruetsch-Chelli: None. A. Manouchehrinia: None. N. Kharlamova: None. M. Naja: None. B. Seitz-Polski: None. D. Isenberg: None. A. Fogdell-Hahn: None. C. Ciurtin: None. E. Jury: None.

Immunogenicity of Rituximab biosimilar GP2013 in chronic inflammatory rheumatic disorders in daily clinical practice

ObjectiveTo study in daily practice the risk of immunogenicity of patients treated with the biosimilar rituximab (RTX) GP2013 used for chronic inflammatory rheumatic disorders. MethodsA prospective monocentric routine care study was carried out between September 2018 and May 2021, including consecutive patients treated with the biosimilar RTX GP2013. Biosamples were taken before each infusion to quantify anti-RTX antibodies (ADAbs) and serum RTX trough levels by ELISA (Lisa Tracker Duo Rituximab, LTR005, Theradiag). Results168 GP2013-treated patients were included (129 who switched from originator RTX and 39 originator RTX naïve). The analysis of 602 samples identified 15 patients (8%) with positive ADAbs including 6 and 9 with transient and persistent ADAbs, respectively. The switch from originator RTX to GP2013 did not increase the risk of immunogenicity, with an incidence rate of 0.8 for 100 patient years. The frequency of persistent ADAs was higher in non-RA patients (5/56, 9% vs. 4/112, 3.5%). Patients with positive persistent ADAbs were more frequently non-caucasian (7/9, 78%, vs. 56/159, 35%, p<0.01) and all had detectable circulating B cells (vs. 40% in ADAb-negative patients, P<0.001). ADAb positivity was not associated with disease activity or RTX discontinuation but patients with ADAb titers >100 ng/mL experienced reduced treatment efficacy or severe infusion-related reaction. ConclusionWithin the study duration, the immunogenicity of GP2013 is a rare event affecting the pharmacodynamics of RTX. Although development of ADAbs had no impact on treatment discontinuation, possible harmful consequences may be observed in patients with high antibody levels.

Patient parameters and response after administration of rituximab in pediatric mature B-cell non-Hodgkin lymphoma.

Mature aggressive B-cell lymphomas are heterogenous malignancies that make up more than half of all diagnosed non-Hodgkin lymphoma in children and adolescents. The overall survival rate increased over the last decades to 80%-90% due to fine tuning of polychemotherapy. However, new therapeutic implications are needed to further increase the overall survival. Current clinical trials analyze the therapeutic effect of rituximab in pediatric patients, while the mechanism of action in vivo is still not fully understood. Effector molecules important for tumor defense were analyzed before and at day 5 after rituximab treatment via flow cytometry. Serum rituximab levels were measured with an ELISA. We evaluated patient parameters that may affect treatment response in relation to rituximab administration and serum rituximab levels. We indeed found a reduction of Fcγ receptor (FcγR) II levels after rituximab treatment in monocyte subtypes, whereas FcγRI expression was significantly increased. Serum levels of proinflammatory marker proteins S100A8/A9 and S100A12 significantly decreased after treatment to normal levels from an overall proinflammatory state before treatment. CD57, perforin, and granzyme B expression decreased after treatment, comprising a less cytolytic natural killer (NK) cell population. The highlighted effects of rituximab treatment on patient's immune response help in understanding the biology behind tumor defense mechanisms and effector function. After subsequent studies, these novel insights might be translated into patient care and could contribute to improve treatment of pediatric patients with mature aggressive B-cell lymphoma.

Rituximab Immunomonitoring Predicts Remission in Membranous Nephropathy.

Primary membranous nephropathy (pMN) is an autoimmune kidney disease and a common cause of nephrotic syndrome in adults. Rituximab is becoming a first line therapy for patients with persistent nephrotic syndrome with proven safety and efficacy, achieving remission in 60%–80% of cases. For the remaining 20%–40% of patients there is an urgent need to identify early biomarkers of resistance to rituximab to adapt therapeutic management. In nephrotic patients, rituximab is found in the blood more transiently than in other autoimmune diseases without proteinuria, due to rituximab wasting in the urine. However, rituximab immunomonitoring is not routinely performed. We evaluated the predictive value of serum rituximab levels in patients with pMN three months after rituximab injection (month-3) on clinical remission rates six months (month-6) and 12 months (month-12) after injection and investigated predictive factors for serum rituximab levels at month-3. Sixty-eight patients treated with rituximab between July 2015 and January 2020 from two French nephrology centers were included. We identified residual rituximab levels at month-3 as a novel early predictor of remission at month-6 (p <0.0001) and month-12 (p = 0.001). Reduced likelihood of remission in patients with undetectable rituximab at month-3 was associated with lower serum albumin and higher anti-PLA2R1 titers at baseline and with lower serum albumin, higher proteinuria, higher CD19+ counts and higher anti-PLA2R1 titers during follow-up. In multivariate analysis, high baseline proteinuria and undetectable rituximab levels at month-3 were independent risk factors for treatment failure at month-6 and high baseline weight and undetectable rituximab levels at month-3 were independent risk factors for treatment failure at month-12. We identified serum albumin at baseline as a predictive factor for serum rituximab levels at month-3. Patients with serum albumin below 22.5 g/L at baseline had an 8.66-fold higher risk of having undetectable rituximab levels at month-3. Therefore, rituximab immunomonitoring in pMN patients treated with rituximab would allow the detection of patients at risk of treatment failure as early as month-3. Studies are needed to assess whether patients with low residual rituximab levels at month-3 may benefit from an early additional course of rituximab.

Clinical Factors Impact on Pharmacokinetics of CT-P10 and Reference Rituximab in Patients with Advanced-Stage Follicular Lymphoma

Background: CT-P10 is the first biosimilar to the innovator rituximab (RTX), which was approved by the European Medicines Agency for all indications for which RTX is approved. Pharmacokinetic equivalence and comparable efficacy was demonstrated in advanced-stage follicular lymphoma patients (Kim WS et al. Lancet Haematol. 2017; S2352-3026(17)) as well as in rheumatoid arthritis (Yoo DH, et al. Arthritis Rheumatol. 2016; 68 (suppl 10)). It is known that there are several factors affecting on pharmacokinetics (PK) of rituximab. However, there are limited data on comparing PK data between a biosimilar of rituximab and RTX with different subgroups in advanced-stage follicular lymphoma (AFL) patients.Objective: This report is to investigate the PK of CT-P10 according to the several relevant clinical factors and to compare with RTX in the post-hoc analysis of randomized controlled trial to compare CT-P10 and RTX in advanced-stage follicular lymphoma (NCT02162771).Methods: A total of 121 patients of PK population were included in this analysis. Fifty nine patients in the CT-P10 group and 62 patients in the RTX group received CT-P10 or RTX (375 mg/m2 i.v) plus CVP (cyclophosphamide, vincristine and prednisone) for every 3 weeks over 8 cycles. Area Under the Concentration-time curve at steady state (AUCtau) and the maximum serum concentration at steady state (CmaxSS) at Cycle 4 and trough serum concentration (Ctrough) at Cycle 4 and Cycle 8 were calculated by standard non-compartmental methods using Phoenix WinNonlin. The parameters were analysed by subgroups of Gender (male vs. female), Eastern Cooperative Oncology Group (ECOG) Performance Status (0 vs. ≥1), Follicular Lymphoma International Prognostic Index (FLIPI) Score (0-2 vs. 3-5) and its component (Age (≥60 vs. <60), Hemoglobin [Hb] (≥120g/L vs. <120g/L), Lactate dehydrogenase [LDH] (>UNL vs. ≤UNL), number of nodal sites (>4 vs. ≤4)), presence of bulky lesion at baseline (≥7cm vs. <7cm) and Complete response (CR)/Complete response unconfirmed (CRu) as best overall response assessed by International Working Group 1999 in the each and combined group of CT-P10 and RTX. Outliers which determined by robust regression outlier testing were excluded in this analysis. For AUCtau, 7 patients (5 in the CT-P10 group and 2 in the RTX group) and among them, 4 patients (2 patients in each group) for CmaxSS were defined as outliers.Result: Higher AUCtau and CmaxSS were observed in patients who are female, under 60 years old, good ECOG Performance Status, low FLIPI score, normal Hb and LDH levels, and non-bulky disease at baseline as shown in Table 1. In patients who achieved CR/CRu as their best overall response during 8 cycles showed higher AUCtau, CmaxSS and Ctrough than in patients who did not achieve CR/CRu response in both CT-P10 and RTX groups. At Cycle 4 and Cycle 8, mean Ctrough in the CR/CRu responder were 72 μg/mL, 108 μg/mL, respectively, and those concentrations are 56 μg/mL, 96 μg/mL in the CR/CRu non-responder. Importantly, there was no statistically significant difference between the CT-P10 and RTX in any of subgroup analyses; confidence interval of the ratio of geometric mean of CT-P10 to RTX contains 1 using Satterthwaite approximation for each comparison.Conclusion: Female gender, younger age (<60), better ECOG Performance Status, lower FLIPI score, LDH and Hb levels in normal range, absence of bulky disease at baseline and better response are factors associated with increased serum rituximab concentration in the combined treatment groups. No statistically significant differences were found between CT-P10 and RTX groups in PK at all subgroup analyses. Therefore, this result shows that the PK of CT-P10 is in accordance with the RTX from historical data and further supports the PK similarity between CT-P10 and RTX. [Display omitted] DisclosuresKim:Celltrion, Inc: Consultancy, Honoraria; Roche: Research Funding; Mundipharma: Research Funding; Novartis: Research Funding; Kyowa-Kirin: Research Funding; Donga: Research Funding; J&J: Research Funding; Takeda: Research Funding. Coiffier:Celltrion, Inc: Consultancy, Honoraria. Buske:Hexal: Honoraria; Celltrion, Inc.: Consultancy, Honoraria; Pfizer: Honoraria; Gilead: Honoraria; Janssen: Honoraria, Research Funding; Roche: Honoraria, Research Funding. Ogura:SymBio: Consultancy, Research Funding; Celgene: Consultancy, Honoraria; Astrazeneca: Honoraria; Takeda: Consultancy, Honoraria; Mundipharma: Consultancy; MeijiSeika Pharma: Consultancy; Celltrion, Inc: Consultancy, Honoraria. Kwak:Celltrion, Inc: Consultancy; InnoLifes: Consultancy, Equity Ownership; Pepromene Bio: Consultancy, Equity Ownership. Lee:Celltrion, Inc: Employment. Bae:Celltrion, Inc: Employment. Kim:Celltrion, Inc: Employment. Kim:Celltrion, Inc: Employment. Lee:Celltrion, Inc: Employment. Kwak:Celltrion, Inc: Employment. Lee:Celltrion, Inc: Employment.

Anti-rituximab antibodies in pediatric steroid-dependent nephrotic syndrome.

Rituximab is a chimeric anti-CD20 monoclonal antibody that induces sustained remission in children with steroid-dependent nephrotic syndrome. However, there is no consensus on the optimal regimen and monitoring of rituximab. In other autoimmune diseases, anti-rituximab antibodies (ARA) have been reported in 10-40% of patients, but their clinical relevance remains unclear. In nephrotic syndrome, data are scarce. We report a single-center retrospective study with immuno- and pharmacological monitoring of rituximab treatment in children with frequent relapsing (FR) or steroid-dependent nephrotic syndrome (SDNS). We analyzed the monthly monitoring of 24 children, receiving a dose of rituximab (375 mg/m2) between December 2017 and April 2018 at the Pediatric Nephrology Department of Robert-Debré hospital, Paris. ARA were detected in 7/24 patients (29%), sometimes after the first infusion of rituximab. ARA were present at baseline in two patients previously treated with rituximab. Both displayed no B-cell depletion. ARA were also reported in 5/22 patients during follow-up, with antibodies always detected in the first month following B-cell recovery. An incomplete CD19+CD20- B-cell depletion at M1 (5-25/mm3) and low serum rituximab levels was predictive of developing ARA. The development of de novo ARA during follow-up was not associated with shorter B-cell depletion. This study shows that ARA are frequent in children with FR/SDNS and that close immuno- and pharmacological monitoring may help personalizing rituximab treatment in patients needing repeated injections.

Immunomonitoring du rituximab appliqué aux maladies auto-immunes : une aide pour la pratique clinique ?

IntroductionImmune monitoring of monoclonal antibodies is a helpful tool in optimizing the management of patients treated with TNF blockers, especially in gastroenterology. In contrast, studies evaluating the interest of such monitoring are lacking for other monoclonal antibodies used in autoimmune diseases, including rituximab despite its widespread use in the field for almost 15 years. Hence, we conducted a study whose goal was to describe the clinical and biological characteristics of all patients who had a rituximab immune monitoring. MethodsAll the clinical, biological and therapeutic data attached to the demands (from 2015 onwards) we received for immune monitoring of rituximab (measurements of rituximab serum levels and anti-rituximab antibodies using the drug-sensitive assay LISA-TRACKER Duo Rituximab®), were retrospectively reviewed. Suspected cases of hypersensitivity and secondary non-response were included. ResultsSeveral medical specialities (nephrology, haematology, neurology, rheumatology, internal medicine) were represented among the 18 records included in the study (out of 23 demands), 10 being suspected cases of hypersensitivity and 8 secondary non-responders. All 6 patients whose symptoms were consistent with the classical presentation of serum sickness, as well as half of the secondary non-responders, were positive for antirituximab antibodies. ConclusionThis detailed real world case study illustrates the potential benefits of rituximab immune monitoring (especially anti-rituximab antibodies) in autoimmune diseases, suggesting it could be helpful in suspected cases of serum sickness, as well as secondary non-response (B-cell non-depletion being an early red flag). Larger and disease-specific studies are warranted to support these findings.

A Phase 1 Multicenter Open-Label Study of Escalating Doses of the Novel Anti-CD20-Targeting Engineered Toxin Body MT-3724 in Subjects with Relapsed or Refractory Mantle Cell Lymphoma

Background: Engineered toxin bodies (ETBs) are comprised of a proprietarily engineered form of Shiga-like toxin A subunit (SLT-A) genetically fused to antibody-like binding domains. ETBs work through novel mechanisms of action and are capable of forcing internalization, self-routing through intracellular compartments to the cytosol, and inducing potent cell-kill via the enzymatic and permanent inactivation by SLT-A of ribosomes. MT-3724 represents a novel ETB modality comprised of an anti-CD20 single-chain variable fragment genetically fused to SLT-A, is capable of efficient internalization once bound to CD20 and can induce potent direct cell-kill via enzymatic ribosome inactivation. In a Phase 1/1b dose escalation/expansion study of MT-3724 monotherapy in subjects with heavily pretreated (including CD20 monoclonal antibodies) relapsed or refractory B-cell non-Hodgkin lymphoma (r/rNHL) the most common grade ≥3 treatment-related adverse events were myalgia and neutropenia (n=3 each). Dose-limiting toxicities (DLTs) were indicative of innate immune response. In subjects with negative rituximab serum concentrations there was a 38% objective response rate (Hamlin et al. ASH 2019). MT-3724 is currently being studied in five ongoing (three actively recruiting, two in development) Phase 2 studies for r/rNHL. This study will evaluate the safety, tolerability, recommended phase 2 dose (RP2D), efficacy, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of MT-3724 in two cohorts of subjects with r/r mantle cell lymphoma (r/rMCL). Study Design and Methods: This phase 1, multicenter, open-label, single arm study will include adults ≥18 yrs with histologically confirmed r/rMCL (histology plus expression of Cyclin D1 in association with CD20 and CD5 or evidence of t(11:14)) who have received ≥2 prior systemic therapies (including anti-CD20 antibody alone or in combination with other agents) and have ≥1 measurable lesion (Lugano criteria). Subjects with CNS involvement or recent treatment with rituximab (within 84 days of study initiation; if received within 12-37 weeks of start of treatment, serum rituximab level must be confirmed to be negative [&amp;lt;500 ng/mL]), obinutuzumab (within 184 days), or ofatumumab (within 88 days) will be excluded. The washout period for these anti-CD20 antibodies, based on published half-life data for these agents, is required due to direct binding competition with MT-3724 for the same CD20 epitope. The primary objective is to evaluate safety and determine the RP2D of MT-3724 in subjects with r/rMCL. Secondary objectives include overall response rate based on the Lugano criteria as determined by the investigators, duration of response, disease control rate, progression-free survival, overall survival, PK, PD, and immunogenicity. An exploratory endpoint will assess if responders become eligible for potentially curative therapy (rate of bridge-to-hematopoietic stem cell transplantation or bridge-to-chimeric antigen receptor T-cell therapy). Subjects will receive escalating doses of MT-3724 as a one hr IV infusion in cycle (C) 1 on Days 1, 4, 8, 11, 18, and 25. In C2+, subjects will receive the highest MT-3724 dose on Days 1, 8, 15, and 22 of a 28-day cycle. Two sequential cohorts will be studied. The study will be initiated with Cohort 1 to evaluate DLTs during the first 42 days of therapy. Subjects will have a dose increase only if they do not experience DLTs and dose reductions will be permitted for treatment-related toxicities. If Cohort 1 is deemed tolerable, Cohort 2 (higher doses) will begin; if Cohort 1 dosing is not tolerable, Cohort 0 (lower doses) will begin (Table 1). A Bayesian optimal interval design will be used to identify the RP2D more accurately (target toxicity rate φ=0.3). Enrollment is anticipated to begin in December 2020. Disclosures Wang: Juno:Consultancy, Research Funding;Dava Oncology:Honoraria;Kite Pharma:Consultancy, Other: Travel, accommodation, expenses, Research Funding;InnoCare:Consultancy;MoreHealth:Consultancy;Targeted Oncology:Honoraria;OMI:Honoraria, Other: Travel, accommodation, expenses;Oncternal:Consultancy, Research Funding;Pulse Biosciences:Consultancy;Molecular Templates:Research Funding;OncLive:Honoraria;Verastem:Research Funding;Acerta Pharma:Research Funding;Celgene:Consultancy, Other: Travel, accommodation, expenses, Research Funding;AstraZeneca:Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding;Janssen:Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding;Pharmacyclics:Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding;BioInvent:Research Funding;Guidepoint Global:Consultancy;VelosBio:Research Funding;Loxo Oncology:Consultancy, Research Funding;Lu Daopei Medical Group:Honoraria;Nobel Insights:Consultancy;Beijing Medical Award Foundation:Honoraria.Burnett:Molecular Templates, Inc.:Current Employment.Strack:Molecular Templates, Inc.:Current Employment.

Abstract PO-63: A phase 2a open-label study of MT-3724, a novel CD20-targeting engineered toxin body, in combination with lenalidomide (LEN) in subjects with relapsed or refractory B-cell non-Hodgkin lymphoma (NHL)

Abstract Background: Engineered toxin bodies (ETBs) are a distinct class of targeted immunotoxins in development by Molecular Templates as anticancer therapeutics. ETBs have a novel mechanism of action that drives a potent and targeted response mediated by antibody-like binding, cellular internalization, and enzymatic ribosomal inhibition via the delivery of a Shiga-like toxin subunit A (SLTA). MT-3724 comprises an anti-CD20 single chain variable fragment genetically fused to SLTA with an approximate molecular weight of 55 kDa and is being developed for the treatment of relapsed or refractory diffuse large B-cell lymphoma (r/rDLBCL). MT-3724 is currently being studied in three ongoing phase 1/2 studies for r/rDLBCL. Methods: MT-3724 is being evaluated in this phase 2a study (NCT03645395) in combination with LEN in adult patients with histologically confirmed, relapsed, or refractory CD20+ B-cell NHL. The primary objectives of this study are to determine the safety and tolerability, including the maximum tolerated dose (MTD), of MT-3724+LEN. Secondary objectives include pharmacokinetics, pharmacodynamics, immunogenicity, and tumor response. The study is being conducted in two parts. Part 1 includes MT-3724 dose escalation over 5 dose cohorts according to the modified 3+3 design and will include up to 24 subjects with CD20+ NHL. Part 2 is designed to assess the safety and tolerability of MT-3724+LEN in the MTD Expansion Cohort, where the dose declared as MTD of MT-3724 in Part 1 will be given in combination with LEN in up to 40 subjects with CD20+ r/rDLBCL. Eligible subjects must have received at least one approved therapy for NHL and must have measurable disease by Lugano criteria. Subjects who have progressed following CAR T-cell therapy, autologous or allogeneic stem cell transplant are also eligible. Serum rituximab level must be negative (&amp;lt;500 ng/mL) at screening because it competes with MT-3724 for binding to CD20. In the first 2 dose cohorts (10 and 25 ug/kg/dose) of Part 1, subjects received MT-3724 IV infusions over 1 hour three times weekly for 2 weeks and LEN (20 mg daily) on Days 1-21 of each 28-day treatment cycle for Cycles 1 and 2. In subsequent cycles, MT-3724 was administered once weekly with continued LEN dosing on Days 1-21. Because of 2 dose-limiting toxicities (grade 2 capillary leak syndrome) in Cohort 2, the dose in Cohort 3 was reduced to 20 ug/kg/dose. The protocol was amended so that in future Cohorts 4 and 5 (25 and 50 ug/kg/dose), MT-3724 will be dosed biweekly for 2 weeks for Cycles 1 and 2 and then once weekly for subsequent cycles. The study is recruiting subjects at multiple study centers. Citation Format: Jason Tache, Deborah A. Katz, Amitabha Mazumder, David Peace, Christine Burnett, Thomas Strack, Elizabeth Bay, Roger Waltzman, Seung Lee. A phase 2a open-label study of MT-3724, a novel CD20-targeting engineered toxin body, in combination with lenalidomide (LEN) in subjects with relapsed or refractory B-cell non-Hodgkin lymphoma (NHL) [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-63.

Evaluation of efficacy and safety of rituximab in combination with mycophenolate mofetil in patients with nonspecific interstitial pneumonia non-responding to a first-line immunosuppressive treatment (EVER-ILD): A double-blind placebo-controlled randomized trial

IntroductionNonspecific interstitial pneumonia (NSIP) are rare but severe diseases, with high mortality and morbidity, with no effective pharmacological treatment allowing for long-term remission, and therefore no clear therapeutic recommendations. Classic immunosuppressants are used as first-line treatment, with only one third of patients being responders and no clear recommendations exist for the choice of the second-line therapy. The EvER-ILD study is the first one to prospectively evaluate the efficacy and safety of rituximab and mycophenolate mofetil (MMF) versus placebo and MMF in a broad range of NSIP patients that did not respond to a first-line therapy. A pharmacokinetic-pharmacodynamic analysis based on rituximab serum concentrations will allow identification of potential factors associated with therapeutic response and/or adverse effects. MethodsEvER-ILD study is a French multicenter, prospective, randomized, double blind, placebo-controlled, superiority trial. Patients with severe and progressive NSIP non-responding to a first line immunosuppressive treatment will be randomized in 2 groups of treatment: one course of rituximab plus 6 months MMF (RTX-MMF group) and one course of placebo plus 6 months MMF (Placebo-MMF group). The primary outcome is the change in Forced Vital Capacity (FVC, % of predicted) from baseline to 6 months. Several clinical, biological, and quality of life secondary outcomes will be measured at 3, 6 and 12 months. A sample size of 122 patients (61 patients per group) would allow to show a point difference between groups in the change of FVC at 6 months, based on a common standard deviation for FVC change of 8% with a power of 90%, alpha 5% two-sided, and anticipating an extreme 10% drop-out rate. Ethics and disseminationThe protocol was approved by the French Research Ethics Committee (CPP Tours Ouest 1 2016-R28) on November 10, 2016, and by the French competent authority (ANSM, reference 160771A-22) on December 1st, 2016. This article refers to protocol V2, dated November 18, 2016. An independent data safety monitoring board will review safety and tolerability data for the duration of the trial. Results will be disseminated via peer reviewed publication and presentation at international conferences. Trial registration numberNCT02990286 (clinicaltrials.gov), EudraCT 2016-003026-16 (European Medicines agency).

A Phase 2 Study of MT-3724 to Evaluate Safety, Pharmacodynamics and Efficacy of MT-3724 for the Treatment of Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma

Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common histologic subtype of non-Hodgkin Lymphoma (NHL) accounting for approximately 30-40% of NHL cases. Approximately 40% of all newly diagnosed DLBCL patients are either refractory or relapsed following initial response to therapy and represent a population with high unmet need for new therapeutic strategies to achieve or regain disease remission. Because of the near ubiquity and persistence of CD20 expression in B-cell malignancies, there is strong rationale to develop compounds with novel mechanisms of action targeting CD20. However, CD20's non-internalizing nature has, to date, leveraged only cytotoxic mechanisms such as antibody-dependent cellular cytotoxicity to which resistance can arise and has impeded the development of agents that internalize a cytotoxic payload. MT-3724 is a novel engineered toxin body designed to overcome this limitation by combining the specific target selectivity of a single chain variable fragment with the lethality of a genetically fused Shiga-like toxin A subunit that facilitates both internalization and cell killing by inactivating ribosomal protein synthesis. MT-3724 has been shown to specifically bind and kill CD20+ malignant human B-cells in vitro (IC50 &lt;1 nM) and in CB17 SCID and PDX mice (Rajagopalan 2016; Huang 2018). As a direct-kill immunotoxin against CD20, MT-3724 has achieved clinical response in subjects with relapsed NHL regardless of acquired resistance to other treatments. Thus, MT-3724 could be a valuable addition to the armamentarium of treating DLBCL. Study Design and Methods: MT-3724 is being evaluated in this Phase 2 study as monotherapy (NCT02361346) in adult subjects with histologically confirmed, relapsed or refractory DLBCL. The primary objective is to determine the efficacy of MT-3724 as monotherapy based on the overall response rate (ORR) by the revised Lugano Classification for Lymphoma adjusted according to immunomodulatory therapy criteria (LYRIC), hereinafter referred to as "revised Lugano Criteria" (Cheson 2014, 2016). Overall response rate is defined as the proportion of subjects with either a complete response or a partial response as determined by independent, blinded central review. Secondary objectives include safety, progression‐free survival, investigator‐assessed ORR, duration of response, and overall survival as well as pharmacokinetics and pharmacodynamics, immunogenicity, and quality of life. To be eligible, patients must have histologically confirmed, relapsed or refractory DLBCL, have received at least 2 standard of care systemic NHL treatment regimens, and have measurable disease according to the revised Lugano criteria. Since rituximab and MT-3724 compete for the same CD20 domain, subjects must have serum rituximab levels &lt; 500 ng/mL before the start of treatment to allow adequate binding of MT-3724. This single arm phase 2 study is being conducted in three stages, where the first two stages will follow the Simon two-stage optimal design [Simon 1989]. Subjects will be enrolled in successive cohorts with each cohort evaluated for efficacy before opening the subsequent cohort, toward a total sample size of 100 subjects. Subjects will receive MT-3724 as an IV infusion over 1 hour on Days 1, 3, 5, 8, 10 and 12 of a 21-day treatment cycle. All subjects will be treated with a 50 µg/kg/dose of MT-3724, which was the recommended Phase 2 dose, as determined in the Phase 1/1b portion of the trial. Sites are open and recruiting in the US, Canada, and Europe. Disclosures Persky: Sandoz: Consultancy; Debiopharm: Other: Member, Independent Data Monitoring Committee; Bayer: Consultancy; Morphosys: Other: Member, Independent Data Monitoring Committee. Musteata:Arensia EM: Other: Principal Investigator; Institute of Oncology: Employment. Strack:Molecular Templates, Inc.: Employment. Burnett:Molecular Templates, Inc.: Employment. Wilson:Molecular Templates, Inc.: Employment. Baetz:Bristol Meyers Squibb: Other: Advisory board; Merck: Other: Advisory board; Gilead: Other: Advisory board; Roche: Other: Advisory board.

A Phase 2a Open-Label Study to Investigate Safety and Tolerability (including the MTD), Efficacy, Pharmacokinetics, Pharmacodynamics and Immunogenicity of MT-3724 in Combination with Lenalidomide in Subjects with Relapsed or Refractory B-Cell Non-Hodgkin Lymphoma

Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common histologic subtype of Non-Hodgkin Lymphoma (NHL) accounting for approximately 30-40% of NHL cases. Approximately 40% of all newly diagnosed DLBCL patients are either refractory or relapsed following initial response to therapy and represent a population with high unmet need for new therapeutic strategies to achieve or regain disease remission. Because of the near ubiquity and persistence of CD20 expression in B-cell malignancies, there is strong rationale to develop compounds with novel mechanisms of action targeting CD20. However, CD20's non-internalizing nature has, to date, leveraged only cytotoxic mechanisms such as antibody-dependent cellular cytotoxicity to which resistance can arise, and has impeded the development of agents that internalize a cytotoxic payload. MT-3724 is a novel engineered toxin body designed to overcome this limitation by combining the specific target selectivity of a single chain variable fragment with the lethality of a genetically fused Shiga-like toxin A subunit that facilitates both internalization and cell killing by inactivating ribosomal protein synthesis. MT-3724 has been shown to specifically bind and kill CD20+ malignant human B-cells in vitro (IC50 &lt;1 nM) and in CB17 SCID and PDX mice (Rajagopalan 2016; Huang 2018). Given MT-3724's unique ability to kill CD20+ cells by ribosomal inhibition, combining MT-3724 with standard cytotoxic chemotherapy and/or immune modulatory agents may provide enhanced benefit in NHL treatment algorithms. To evaluate this possibility, MT-3724 was tested in combination with Lenalidomide (LEN) to assess additive, synergistic or antagonistic effects in CD20-positive Daudi cell lines. This combination significantly (Ki = 0.40) potentiated cytotoxicity. Based on these results and the known safety profile of MT-3724, LEN was chosen as a combination agent to maximize anti-tumor effects in patients with B-cell NHL while minimizing overlapping toxicity risks. Study Design and Methods: MT-3724 is being evaluated in this Phase 2a study (NCT03645395) in combination with LEN in adult patients with histologically confirmed, relapsed or refractory NHL. The primary objective of this study is to determine the safety and tolerability, including the maximum tolerated dose (MTD), of MT-3724 + LEN. Secondary objectives include pharmacokinetics (PK), pharmacodynamics (PD), immunogenicity and tumor response in subjects treated with MT-3724 + LEN. The study will be conducted in two parts. Part 1 will include MT-3724 dose escalation according to the modified 3+3 design to identify the MTD of MT-3724 in combination with standard doses of LEN and will include up to 24 subjects with histologically confirmed NHL. Part 2 is designed to confirm the safety and tolerability of MT-3724 + LEN in the MTD Expansion Cohort, where the dose declared as MTD of MT-3724 in Part 1 would be given in combination with LEN in up to 40 subjects with CD20+ DLBCL. In addition, the PK, PD, immunogenicity and tumor response of MT-3724 + LEN will be evaluated in Part 2. Eligible subjects will have histologically confirmed, relapsed or refractory CD20+ B-cell NHL that, in the investigator's opinion, could benefit from MT-3724 + LEN therapy. Patients must have received all approved therapies for NHL that are applicable for the patient in the opinion of the treating physician and must have measurable disease by Lugano Classification for NHL. Subjects that are refractory to treatment are eligible, as well as patients who have progressed following CAR T-cell therapy, autologous- or allogeneic stem cell transplant. Serum rituximab level must be negative (&lt;500 ng/mL) at screening. Subjects will receive MT-3724 IV infusions over 1 hour on Days 1, 3, 5, 8, 10 and 12 and will receive LEN on days 1-21 of each 28-day treatment cycle during cycles 1 and 2. Thereafter, MT-3724 will be administered weekly on Days 1, 8, 15, and 22 of each cycle with continued LEN dosing on Days 1-21 for cycles 3 to 6. The study is currently ongoing. Eligible subjects will be identified and treated through competitive enrollment at multiple study centers in North America. Disclosures Burnett: Molecular Templates, Inc.: Employment. Gordon:Abbott: Equity Ownership; Abbvie: Equity Ownership. Strack:Molecular Templates, Inc.: Employment. Lehner:Molecular Templates, Inc.: Employment. Higgins:Molecular Templates, Inc.: Employment, Equity Ownership.

A Phase 2a Open-Label Study to Investigate Safety and Tolerability (including the MTD), Efficacy, Pharmacokinetics, Pharmacodynamics and Immunogenicity of MT-3724 in Combination with Gemcitabine and Oxaliplatin in Subjects with Relapsed or Refractory B-Cell Non-Hodgkin Lymphoma

Introduction: Approximately 40% of all newly diagnosed Diffuse large B-cell lymphoma (DLBCL) patients are either refractory or relapsed following initial therapy, and represent a population with high unmet need for new therapeutic strategies to achieve or regain disease remission. Because of the near ubiquity and persistence of CD20 expression in B-cell malignancies, there is strong rationale to develop compounds with novel mechanisms of action (MOA) targeting CD20. However, CD20's non-internalizing nature has, to date, leveraged only cytotoxic mechanisms such as antibody-dependent cellular cytotoxicity to which resistance can arise. MT-3724 is a novel engineered toxin body designed to overcome this limitation by combining the specific target selectivity of a single chain variable fragment with the lethality of a genetically fused Shiga-like toxin A subunit that facilitates both internalization and cell killing by inactivating ribosomal protein synthesis. MT-3724 has been shown to specifically bind and kill CD20+ malignant human B-cells in vitro (IC50 &lt;1 nM) and in CB17 SCID and PDX mice (Rajagopalan 2016; Huang 2018). MT-3724 has been studied in a dose-escalation monotherapy trial (NCT02361346) and has achieved clinical response as monotherapy in subjects with relapsed NHL regardless of acquired resistance to other treatments. Given MT-3724's unique ability to kill CD20+ cells by ribosomal inhibition, combining MT-3724 with standard cytotoxic chemotherapy may provide enhanced benefit in NHL treatment. To evaluate this possibility, MT-3724 was tested in combination with selected cytotoxic compounds including gemcitabine and cisplatin in various CD20+ cell lines (Raji, SU-DHL-4, Daudi, HBL-1). MT-3724 showed synergistic cytotoxic effects with gemcitabine and mixed additive/synergistic or synergistic effects with cisplatin in all 4 cell lines. Based on these results and the known safety profile of MT-3724, gemcitabine and oxaliplatin (GEMOX) were chosen as combination agents to maximize anti-tumor effects in patients with CD20+ B-cell NHL while minimizing overlapping toxicity risks. Study Design and Methods: MT-3724 is being evaluated in this Phase 2a study (NCT03488251) in combination with GEMOX, in adult subjects with histologically confirmed, relapsed or refractory CD20+ B-cell NHL. The primary objective of this study is to determine the safety and tolerability including the maximum tolerated dose (MTD) of MT-3724+GEMOX. Secondary objectives include the assessment of pharmacokinetics (PK), pharmacodynamics (PD), immunogenicity and tumor response. The study will be conducted in two parts. Part 1 will include MT-3724 dose escalation according to a modified 3+3 design to identify the MTD of MT-3724 in combination with GEMOX and will include up to 24 subjects with histologically confirmed NHL. All subtypes of B-cell NHL are eligible for Part 1. Part 2 is designed to confirm the safety and tolerability of MT-3724 in the MTD Expansion Cohort, MT-3724 would be given in combination with GEMOX in up to 40 subjects with CD20+ DLBCL. In addition, the PK, PD, immunogenicity and tumor response of MT-3724 in combination with GEMOX. Eligible subjects will have histologically confirmed, relapsed or refractory CD20+ B-cell NHL that could benefit from MT-3724+GEMOX therapy. Subjects must have received all approved therapies for NHL that are applicable for the subject in the opinion of the treating physician and must have measurable disease by Lugano criteria. Subjects that are refractory to treatment are eligible, as well as patients who have progressed following CAR T-cell therapy, autologous- or allogeneic stem cell transplant. Residual rituximab causes MT-3724 to be ineffective as per the previous study experience therefore serum rituximab level must be &lt;500 ng/mL at screening so that the MOA of MT-3724 is not inhibited. Subjects will receive MT-3724 IV infusions over 1 hour in 3 successive dose cohorts (at 10, 20, and 50-µg/kg/dose) on Days 1, 3, 5, 8, 10 and 12 and will receive GEMOX (GEM 1000 mg/m2, and OX 100mg/m2) on Days 2 and 16 of each 28-day treatment cycle during cycles 1 and 2. Thereafter, MT-3724 will be administered weekly on Days 1, 8, 15, and 22 of each cycle with GEMOX on Days 2 and 16 for cycles 3 and 4. The study is currently ongoing. Eligible subjects will be recruited across multiple sites in North America; additional sites in other global regions may be included if necessary. Disclosures Burnett: Molecular Templates, Inc.: Employment. Strack:Molecular Templates, Inc.: Employment. Lehner:Molecular Templates, Inc.: Employment. Higgins:Molecular Templates, Inc.: Employment, Equity Ownership. Katz:Dova: Consultancy; Stemline: Speakers Bureau.