Humanized anti-CD33 Antibody Research Articles

Fractionated Gemtuzumab Ozogamicin in Association with High Dose Chemotherapy: A Bridge to Allogeneic Stem Cell Transplantation in Refractory and Relapsed Acute Myeloid Leukemia?

Primary refractory and relapsed acute myeloid leukemia (PR-AML) are associated with a very dismal prognosis. Allogeneic stem cell transplantation (allo-HSCT) is the only salvage option with curative potential in this situation. Since the disease status at transplant remains one of the main prognostic factors it is critical to reach complete remission (CR) at this stage. Gemtuzumab ozogamicin (GO) is a humanized anti-CD33 antibody conjugated with the cytotoxic antitumor antibiotic calicheamicin, whose results in salvage therapy remain debated. Here, we tested the combination of fractionated GO (fGO, 3mg/m² day 1, 3 and 7) with intensive chemotherapy, mainly consisting in cytarabine (1g/m² twice daily in day 1-5) and mitoxantrone (12mg/m² day 1, 2 and 3) (MYLODAM protocol).The present single center retrospective analysis aimed to assess the outcomes of PR-AML patients treated by GO associated with intensive chemotherapy. The following prognostic factors were recorded: age, cytogenetics, molecular data, blood cell counts, organ failure, and duration of first complete remission. The primary endpoints of this study were the efficacy and safety profile of this salvage strategy. We also attempted to define predictive factors to survival and response after salvage. The ELN 2017 classification was used for prognostic assessment and definition of response.Fifty-eight patients were included. Patients' characteristics are shown in Table 1. Median age at salvage was 56 (range 16-74) years. Thirty-six (62%) received MYLODAM and 14 (24%) had fGO combined with cytarabine (1g/m² twice daily in day 1-5) and daunorubicine (60mg/m² day 1,2 and 3). Forty-six patients (79%) were monitored by molecular biology for minimal residual disease (MRD). The overall response rate was 67% (17% CRi, 17% CR and 33% CRMRD-). Median leukemia-free survival (LFS) and overall survival (OS) were 13.5 months (95% CI: 6-21) and 50 months (95% CI: 11-90) respectively (Figs 1 and 2). LFS and OS at 2 years were 36% (95%CI: 23 - 49) and 54% (95%CI: 39 - 68), respectively. Therapy-related mortality was observed in 7% (n=4) of the patients caused by 2 sepsis and 1 hemorrhagic syndrome. Three patients developed a sinusoidal obstruction syndrome (SOS), 2 of moderate and 1 of severe grade leading to death. In multivariate analysis, LFS was associated with FLT3 mutations (HR=0.33, 95%CI: 0.11-0.97) and adverse ELN risk group (HR=3.5, 95% CI: 1.5-8.2).On the whole, 28 (48%) patients could proceed to allo-HSCT. For this subgroup, LFS and OS at 2 years were 57 % (95% CI: 36 - 77) and 69 % (95% CI: 49 - 89), respectively. The cumulative incidences of grade II-IV acute GVHD and chronic GVHD were 40% and 45.5%, respectively. At 2 years, non-relapse mortality was 16 % (95% CI: 8.5 - 33.3). Despite policy of our center to prevent SOS in this high risk patients by using Defibrotide prophylaxis, 5 SOS syndromes occurred during allogenic transplant with 3 moderate and 2 very severe forms, with no SOS related-deaths.In summary, we conclude that, in a “real-life” setting, fGO associated with intensive chemotherapy (mainly MYLODAM) can be an effective and safe salvage regimen in patients with PR-AML, allowing in two-thirds of the cases a response achievement, and in half of the patients a bridge to allo-HSCT with encouraging outcomes. [Display omitted] DisclosuresMohty:Sanofi: Honoraria, Speakers Bureau.

Inotuzumab Ozogamicin (InO) Vs Standard of Care (SC) in Patients with Relapsed/Refractory (R/R) Acute Lymphoblastic Leukemia (ALL): Long-Term Results of the Phase 3 INO-VATE Study

Inotuzumab Ozogamicin (InO) Vs Standard of Care (SC) in Patients with Relapsed/Refractory (R/R) Acute Lymphoblastic Leukemia (ALL): Long-Term Results of the Phase 3 INO-VATE Study

Duration of response to loncastuximab tesirine in relapsed/refractory diffuse large B-cell lymphoma by demographic and clinical characteristics: Subgroup analyses from LOTIS 2.

7546 Background: Outcomes for patients with refractory/relapsed diffuse large B-cell lymphoma (R/R DLBCL) are poor, particularly for those with high-risk clinical characteristics. There remains an unmet need for new treatment options for these patients. Loncastuximab tesirine (Lonca) is an antibody-drug conjugate comprising a humanized anti-CD19 antibody conjugated to a potent pyrrolobenzodiazepine dimer toxin. LOTIS 2 was a pivotal Phase 2 study that demonstrated substantial single-agent anti-cancer activity of Lonca in patients with R/R DLBCL. Efficacy and safety data were presented at ASH 2020 (Caimi et al, ASH 2020; abstract 1183). Here we present subgroup analyses of duration of response (DoR) to Lonca by demographic and clinical characteristics. Methods: Adult patients with R/R DLBCL who had received ≥2 prior therapies were enrolled in this Phase 2, multicenter, single-arm, open-label study of single-agent Lonca (150 µg/kg every 3 weeks for 2 doses, followed by 75 µg/kg thereafter for up to 1 year). The primary analysis has previously been reported, with a primary endpoint of overall response rate (ORR). Patients are being followed-up every 12 weeks for up to 3 years. DoR was a key secondary efficacy endpoint, defined as time from the first documentation of response (central review) to disease progression or death. We analyzed pre-specified demographic and clinical characteristic subgroups for DoR. Results: As of data cut-off (August 6, 2020), ORR in the total population (N = 145) was 48.3% (24.8% had complete response [CR] and 23.4% had partial response [PR]). Median DoR (mDoR) for the 70 responders was 12.58 months. mDoR for patients with CR and PR was 13.37 months and 5.68 months, respectively. Overall, subgroups with high-risk characteristics for poor prognosis had a DoR comparable to the whole study population. mDoR for patients with double-/triple-hit DLBCL was 13.37 months, with advanced stage disease was 12.58 months, and with transformed disease was 12.58 months. The mDoR for older patients was longer than for younger patients (≥75 years, 13.37 months; 65 to < 75 years, 12.58 months; < 65 years, 9.26 months). Patients with DLBCL refractory (defined as no response to therapy) to first-line, most recent line, and all prior lines of therapy had mDoRs of 9.63 months, 9.26 months, and 9.63 months, respectively. Conclusions: Durable responses were observed with the recommended Phase 2 dose regimen of Lonca in heavily pre-treated patients and those at high risk of poor prognosis, including older patients and those with double-/triple-hit, advanced stage, transformed, and primary refractory DLBCL. Updated DoR data will be presented at the meeting. Clinical trial information: NCT03589469.

The human anti-CD40 agonist antibody mitazalimab (ADC-1013; JNJ-64457107) activates antigen-presenting cells, improves expansion of antigen-specific T cells, and enhances anti-tumor efficacy of a model cancer vaccine in vivo

Non-responders to checkpoint inhibitors generally have low tumor T cell infiltration and could benefit from immunotherapy that activates dendritic cells, with priming of tumor-reactive T cells as a result. Such therapies may be augmented by providing tumor antigen in the form of cancer vaccines. Our aim was to study the effects of mitazalimab (ADC-1013; JNJ-64457107), a human anti-CD40 agonist IgG1 antibody, on activation of antigen-presenting cells, and how this influences the priming and anti-tumor potential of antigen-specific T cells, in mice transgenic for human CD40. Mitazalimab activated splenic CD11c+ MHCII+ dendritic cells and CD19+ MHCII+ B cells within 6 h, with a return to baseline within 1 week. This was associated with a dose-dependent release of proinflammatory cytokines in the blood, including IP-10, MIP-1α and TNF-α. Mitazalimab administered at different dose regimens with ovalbumin protein showed that repeated dosing expanded ovalbumin peptide (SIINFEKL)-specific CD8+ T cells and increased the frequency of activated ICOS+ T cells and CD44hi CD62L− effector memory T cells in the spleen. Mitazalimab prolonged survival of mice bearing MB49 bladder carcinoma tumors and increased the frequency of activated granzyme B+ CD8+ T cells in the tumor. In the ovalbumin-transfected tumor E.G7-OVA lymphoma, mitazalimab administered with either ovalbumin protein or SIINFEKL peptide prolonged the survival of E.G7-OVA tumor-bearing mice, as prophylactic and therapeutic treatment. Thus, mitazalimab activates antigen-presenting cells, which improves expansion and activation of antigen-specific T cells and enhances the anti-tumor efficacy of a model cancer vaccine.

A humanized CD3ε-knock-in mouse model for pre-clinical testing of anti-human CD3 therapy.

Pre-clinical murine models are critical for translating drug candidates from the bench to the bedside. There is interest in better understanding how anti-human CD3 therapy works based on recent longitudinal studies of short-term administration. Although several models have been created in this pursuit, each have their own advantages and disadvantages in Type-1 diabetes. In this study, we report a murine genetic knock-in model which expresses both a murine and a humanized-CD3ε-exon, rendering it sensitive to manipulation with anti-human CD3. These huCD3εHET mice are viable and display no gross abnormalities. Specifically, thymocyte development and T cell peripheral homeostasis is unaffected. We tested immune functionality of these mice by immunizing them with T cell-dependent antigens and no differences in antibody titers compared to wild type mice were recorded. Finally, we performed a graft-vs-host disease model that is driven by effector T cell responses and observed a wasting disease upon transfer of huCD3εHET T cells. Our results show a viable humanized CD3 murine model that develops normally, is functionally engaged by anti-human CD3 and can instruct on pre-clinical tests of anti-human CD3 antibodies.

Abstract PS17-06: Humanized anti-CD47 monoclonal antibody magrolimab (Hu5F9-G4) plus trastuzumab potentiates antibody-dependent cellular phagocytosis (ADCP), and cooperate to inhibit human HER2+ breast cancer (BC) xenografts growth in vivo

Abstract Background: Cancer cells overexpress CD47 to evade phagocytic programmed cell removal (PrCR) by macrophages of the innate immune system. Blocking CD47 using magrolimab (Hu5F9-G4), an anti-CD47 humanized monoclonal antibody, works via preventing CD47 signaling via macrophage Sirpα to allow PrCR, as well as using an Fc-receptor mechanism to encourage macrophage-mediated phagocytosis. We have previously shown that magrolimab in combination with tumor-targeting antibodies (e.g. rituximab) further enhances magrolimab’s anti-cancer effects in preclinical models and in the clinic [Advani R, et al. N Engl J Med 2018; 379:1711-1721]. We therefore hypothesized that magrolimab combined with anti-HER2 monoclonal antibody would synergize to promote ADCP in vitro, and HER2+ breast xenograft growth inhibition in vivo. Methods: To test this hypothesis, 1 x 105 CFSE-labeled HER2+ BT474 and SKBR3 cells were plated in 96-well ultra-low attachment plates in serum-free media. Isotype control antibody, trastuzumab, magrolimab, and trastuzumab + magrolimab combination (10µg/ml) were added, and allowed to incubate at 37°C for 30 mins. 5 x 104 human macrophages were then added each well, and were co-cultured for 2 hours. Human macrophages were stained with anti-CD11b, and phagocytosis was determined as the percentage of cells that were CD11b+ and CSFE+ using a BD LSR Fortessa Analyzer. For in vivo tumor xenograft growth kinetics, GFP+/Luciferase+ BT474 cells (1X105) were implanted with 25% Matrigel into the mammary fat pads of 4-8 week-old NOD scid gamma (NSG) female mice. Twenty-five days after engraftment, trastuzumab (100ug) was administered via intraperitoneal (IP) injection weekly, magrolimab 250ug IP was administered every other day, and PBS control was administered IP at 100uL once weekly, and tumor growth was monitored for 17 weeks by bioluminescence (IVIS) after D-luciferin injection, and quantified using Image 4.0. Results: SKBR3 and BT474 parental and Trastuzumab-resistant lines demonstrated significantly increased susceptibility to ADCP when opsonized with combination treatment of trastuzumab + magrolimab (15.34, 95%CI= 12.2 to 16.6) compared to trastuzumab (9.66, 95%CI=1.861 to 7.7579, p = 0.034), or magrolimab alone (10.23, 95%CI=1.254 to 7.037, p = 0.0083). Treatment with magrolimab maintained tumor burden within the starting range but further progressed upon treatment cessation (2.09x109, CI95% = 2801531434 to 15343840962, p = 0.0006). Trastuzumab treatment resulted in lower starting range tumor burden, but also demonstrated progression once treatment was stopped (1.72x106, CI95% = -695208489 to -86402608, p = 0.0052). However, in the combinatorial treatment arm, tumors were significantly below the starting range and did not show signs of tumor progression within the 10-week non-treatment period (8.51x105, CI95%= -311406356 to -66914472, p = < 0.0001). Conclusion: We conclude magrolimab plus trastuzumab cooperate to inhibit HER2+ xenograft growth in vivo, and that treatment effect persists even after treatment is stopped. Our ex vivo data suggests one mechanism to explain the observed tumor growth inhibition is increased susceptibility to ADCP when HER2+ tumors are opsonized by the combination of trastuzumab + magrolimab. Future clinical translation of this combination is warranted. Citation Format: Rosalynd Upton, Dongdong Feng, Allison M Banuelos, Tanuka Biswas, Stephen Willingham, Kevin S Kao, Kelly McKenna, Benyamin Rosenthal, Michal C Tal, Jens-Peter Volkmer, Mark D Pegram, Irving L Weissman. Humanized anti-CD47 monoclonal antibody magrolimab (Hu5F9-G4) plus trastuzumab potentiates antibody-dependent cellular phagocytosis (ADCP), and cooperate to inhibit human HER2+ breast cancer (BC) xenografts growth in vivo [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-06.

The use of tafasitamab in diffuse large B-cell lymphoma

Patients who relapse or are refractory after first-line therapy for diffuse large B-cell lymphoma (DLBCL) frequently have poor prognoses, especially when they are not candidates for autologous stem cell transplant (ASCT). Tafasitamab is a humanized monoclonal anti-CD19 antibody that has recently been approved by the FDA in combination with lenalidomide for the treatment of relapsed/refractory (R/R) DLBCL in patients who are not eligible for ASCT. Tafasitamab has an Fc region which has been modified to have an increased affinity for Fcγ receptors, to potentiate antibody-dependent cellular cytotoxicity and antibody-dependent cell-mediated phagocytosis. Here, we review the development, mode of action and clinical data for tafasitamab in combination with lenalidomide in R/R DLBCL, and discuss the various ways in which this novel antibody could be utilized in the treatment sequence to improve clinical outcomes for patients with DLBCL.

B cell targeting therapies in MS patients during the SARS-CoV-2 pandemic - when immunosuppression meets infection?

Research into the mechanisms of autoimmune demyelination have highlighted B cells in this process. Therapies targeting this population were a recent addition to the multiple sclerosis (MS) drugs portfolio. The SARS-CoV-2 pandemic and the risk of severe COVID-19 have challenged the safety of B cell depletion in MS patients. Selective depletion of B cells by monoclonal antibodies as monotherapy in MS has been shown to profoundly suppress disease activity among relapsing-remitting MS patients. Furthermore ocrelizumab, a humanised anti-CD20 monoclonal antibody, was the first licensed therapy in primary progressive MS. Based on the concept of the role of B cells in MS, many therapeutic approaches are emerging as novel ways to treat autoimmune demyelination. However, during the SARS-CoV-2 pandemic, a conservative approach toward limiting immune suppression in MS patients has been proposed. Emerging evidence does not support the notion of increased susceptibility among MS patients to the SARS-CoV-2 infection, or any predisposition toward greater severity of COVID-19. This also does not appear to be the case for MS patients undergoing B cell depletion therapies. Thus, any decision to withhold immune suppression in MS patients during the SARS-CoV-2 pandemic is probably incorrect. MS therapeutic decision-making should focus on the danger of poorly controlled autoimmune demyelination rather than perceived risks from COVID-19. The current pandemic highlights the need to develop more selective and safer methods of immunomodulation in MS. B cells represent several functionally different populations. Further research into the different role of these cells during autoimmune demyelination should yield better, safer strategies to control the encephalitogenic process.

Efficacy and Safety of Loncastuximab Tesirine (ADCT-402) in Relapsed/Refractory Diffuse Large B-Cell Lymphoma

Efficacy and Safety of Loncastuximab Tesirine (ADCT-402) in Relapsed/Refractory Diffuse Large B-Cell Lymphoma

Safety and Antitumor Activity Study Evaluating Loncastuximab Tesirine and Rituximab Versus Immunochemotherapy in Diffuse Large B-Cell Lymphoma

Introduction: Patients with diffuse large B-cell lymphoma (DLBCL) who fail immunochemotherapy (IC) and are unsuitable for autologous stem cell transplantation (ASCT) and those who relapse shortly after ASCT have extremely poor prognosis and need additional treatment options. Loncastuximab tesirine (Lonca) is an antibody-drug conjugate (ADC) composed of a humanized anti-CD19 antibody conjugated to a pyrrolobenzodiazepine dimer toxin. In a Phase 2 study (NCT03589469), Lonca demonstrated single-agent antitumor activity with manageable toxicity in patients with relapsed/refractory (R/R) DLBCL. Rituximab is a CD20-targeting monoclonal antibody used in front-line IC for DLBCL and in salvage regimens, such as rituximab/gemcitabine/oxaliplatin (R-GemOx). The addition of rituximab to a CD19-targeting pyrrolobenzodiazepine ADC appears to prolong tumor control in preclinical studies, providing the rationale for evaluating Lonca combined with rituximab (Lonca-R) as a treatment for R/R DLBCL. Study Design and Methods: This is a Phase 3, randomized, open-label, 2-part, 2-arm, multicenter study of Lonca-R versus standard IC in patients with R/R DLBCL (NCT04384484). Part 1 is a nonrandomized safety run-in with Lonca-R. The toxicity of Lonca-R will be compared with previous single-agent Lonca safety data after 20 patients have completed Cycle 1 in Part 1. Provided no significant increase in toxicity is observed, Part 2 will be initiated. Part 2 is a randomized study of Lonca-R versus R-GemOx (Figure 1). Key inclusion and exclusion criteria are reported in Table 1. The primary objective of Part 2 is to evaluate the efficacy of Lonca-R versus R-GemOx, using progression-free survival (PFS) as the primary endpoint. PFS will be defined as the time between randomization and first documentation of recurrence, disease progression or death (central review) and the primary analysis will compare PFS between treatment arms using stratified log-rank testing. Secondary objectives include evaluation of safety, pharmacokinetics, and immunogenicity of the combination, in addition to the impact of treatment on symptoms, patient-reported outcomes and patients' overall health. In Part 1 and in the Lonca-R arm of Part 2, patients will receive intravenous (iv) Lonca at 150 µg/kg on day 1 of each 21-day cycle for 2 cycles, then at 75 µg/kg on day 1 for up to 6 additional cycles. Rituximab 375 mg/m2 iv will be administered subsequent to Lonca infusion on day 1 of each cycle. Patients treated with Lonca-R will also be given dexamethasone 4 mg (oral, twice a day), where not contraindicated, on the day before, the day of, and the day after Lonca-R infusion. In the R-GemOx arm, patients will receive rituximab 375 mg/m2, gemcitabine 1000 mg/m2, and oxaliplatin 100 mg/m2 iv on day 1 of each 14-day cycle up to a total of 8 cycles. Patients will receive premedication and supportive care according to the respective prescribing information for rituximab, gemcitabine, and oxaliplatin. The trial is planned to open in Q3/Q4 2020, and target enrollment is 350 patients. Funding: This study is sponsored by ADC Therapeutics SA; https://clinicaltrials.gov/ct2/show/NCT04384484. Disclosures Linhares: Jazz Pharmaceuticals: Consultancy; ADC Therapeutics, Verastem Oncology, Bristol Myers-Squibb (Juno), AstraZeneca: Research Funding; Miami Cancer Institute, Baptist Health South Florida: Current Employment. Gandhi:TG Therapeutics (Advisory board), GlaxoSmithKline (Advisory board): Membership on an entity's Board of Directors or advisory committees. Adeleye:ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ungar:ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hamadani:ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme, AstraZeneca: Speakers Bureau; Janssen R&D; Incyte Corporation; ADC Therapeutics; Celgene Corporation; Pharmacyclics, Omeros, AbGenomics, Verastem, TeneoBio: Consultancy; Takeda Pharmaceutical Company; Spectrum Pharmaceuticals; Astellas Pharma: Research Funding. OffLabel Disclosure: Rituximab is licensed for treatment of NHL but is being used in combination with an unlicensed drug (loncastuximab tesirine) in this study

Long-term cognitive impairments following recovery in the patients with immune-mediated thrombotic thrombocytopenic purpura.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a life-threatening thrombotic disorder caused by the production of anti-ADAMTS13 antibodies that leads to a severe decline in ADAMTS13 activity. Without proper treatment, it has a mortality rate of approximately 90%.1 A combination regimen of plasma exchange (PEX), using fresh frozen plasma and corticosteroids, has reduced iTTP-associated mortality to below 20%. The emerging therapeutic options, including anti-human CD20 antibody, rituximab and anti-von Willebrand factor (VWF) A1 domain nanobody, and caplacizumab,2 have demonstrated efficacy in the management of acute iTTP. Although some iTTP patients may relapse and receive additional treatments, the overall treatment outcomes for iTTP have improved significantly in the past three decades––from an unknown fetal disease to a well-understood and treatable autoimmune disorder. In contrast, there are multiple, unexpected, long-term comorbidities related to iTTP that have gradually emerged, including the risks of developing systemic lupus erythematosus, hypertension, diabetes, renal impairment and neuropsychiatric disorders (including cognitive disorders and depression).3-5 Among these comorbidities, identifying cognitive symptoms can be challenging to physicians unless they pay careful attention. Alwan et al. investigated cerebral magnetic resonance imaging (MRI) findings in 131 patients (119 iTTP and 12 congenital TTP) who complained of various neurological symptoms, ranging from persistent headache to seizures, during acute episodes of TTP. The data showed that 56% of patients had abnormal image findings, and those with severe neurological symptoms were more likely to have abnormal MRI findings than those with headaches only (80% vs. 18%, respectively). All the patients with iTTP received a combination of PEX and immunosuppressors (steroids and rituximab), while none received caplacizumab. Subsequent extensive neuropsychology assessments of 35 survivors at a median of 33 months after the acute episode revealed the following conditions: 63% had deteriorated executive function, 57% had memory impairment, and 43% had intellectual impairment (verbal and non-verbal). The MRI findings at the acute phase and neuropsychology assessments at the remission phase revealed a link between hyper-intense white matter lesions, mainly seen in the frontal and supratentorial regions in MRI images, and severe cognitive impairment. To the best of my knowledge, this is the first study to show the relationship between abnormal findings in cerebral MRI and cognitive impairments, identified by detailed neuropsychological assessments. Intriguingly, depression was not associated with the cerebral abnormalities that could be detected by MRI in the acute phase of the disease. The results of this study highlight the importance of recognising cognitive impairments as long-term comorbidities, suggesting that we should carefully follow not only haemolytic markers, but also the neuropsychiatric status in iTTP patients with cerebral MRI abnormalities at the first presentation. Previous studies have shown that a higher degree of memory impairment in remission detected in patients with neurological events during acute iTTP,5 and the symptoms associated with cognitive impairment did not improve over time.3 Hence, attention is focussed on understanding whether VWF blockage with caplacizumab in the very early phase of the disease could avoid ischaemic cell damage in the brain, preventing cognitive impairment in remission. In fact, standard therapy with caplacizumab could reduce thrombotic events in newly diagnosed iTTP significantly as has been shown in the phase III study.2 In addition, it has been previously reported that an impaired Glasgow Coma Scale at the acute phase indicates poor outcome.6 If caplacizumab could be effective in treating both acute neurological symptoms and cognitive impairments during long-term follow-up, then it might be a reasonable option for iTTP patients who develop severe neurological abnormalities, such as coma, seizure and other stroke-like symptoms, and have abnormal findings on cerebral MRI in the first presentation. When we see patients with iTTP in remission at the outpatient clinic, an extensive assessment from multiple points of view, including neurological and psychiatric evaluations, will help to indicate long-term comorbidity. The author declares no conflict of interest.

ABCL-170: Long-Term Outcomes from the Phase II L-MIND Study of Tafasitamab (MOR208) Plus Lenalidomide in Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma

Context: Tafasitamab, a humanized anti-CD19 antibody, has demonstrated encouraging activity with durable responses with lenalidomide (LEN) in autologous stem cell transplant (ASCT)-ineligible patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) in the Phase II L-MIND study ( NCT02399085 ). In the primary analysis (cut-off November 30, 2018), the objective response rate (ORR) was 60.0%, median duration of response (DOR) was 21.7 months and median follow-up was 17.3 months. Objective: To report the long-term clinical efficacy and safety of tafasitamab + LEN in the L-MIND study after one additional year of follow-up (cut-off November 30, 2019). Design & Setting: An open-label, single-arm, Phase II study. Patients: Aged ≥18 years with R/R DLBCL (1–3 prior systemic therapies, including ≥1 CD20-targeting regimen), an ECOG performance status 0–2, and ASCT ineligible. Interventions: Tafasitamab (12 mg/kg intravenously, 28-day cycles) was administered once weekly during Cycles 1–3 with a loading dose on Cycle 1 Day 4, then every 2 weeks during Cycles 4–12. LEN (25 mg orally) was self-administered on Days 1–21 of Cycles 1–12. After Cycle 12, progression-free patients received tafasitamab every 2 weeks until disease progression. Main Outcome Measure(s): The primary endpoint was ORR (independent review committee). Secondary endpoints included DOR, progression-free survival (PFS), overall survival (OS), and safety. Results: In this long-term analysis, 80 of 81 enrolled patients received tafasitamab + LEN and were included in the efficacy analysis. ORR was 58.8% (n=47/80); 41.3% (n=33/80) with complete response; and 17.5% (n=14/80) with partial response. Median DOR was 34.6 months (95% confidence interval [CI]: 26.1–not reached [NR]). Median OS was 31.6 months (95% CI: 18.3–NR) with a median follow-up of 31.8 months. Median PFS was 16.2 months (95% CI: 6.3–NR) with a median follow-up of 22.6 months. No unexpected toxicities were reported. Conclusions: After a minimum of two years' follow-up, outcomes are consistent with the primary analysis and confirm the durability of response and meaningful OS of tafasitamab + LEN followed by tafasitamab in ASCT-ineligible patients with R/R DLBCL. Along with an acceptable safety profile, these data support a clinically relevant benefit with this immunological combination.

ABCL-135: RE-MIND: A Comparison of Tafasitamab (MOR208) + Lenalidomide (L-MIND) Versus Lenalidomide Monotherapy (Real-World Data) in Transplant-Ineligible Patients with Relapsed/Refractory Diffuse Large B-Cell Lymphoma

Context: Tafasitamab (MOR208), a humanized anti-CD19 antibody, combined with lenalidomide (LEN) showed encouraging results in the Phase II, single-arm L-MIND study ( NCT02399085 ) of patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) ineligible for autologous stem cell transplantation (ASCT). Objective: To generate a LEN monotherapy matched comparator cohort for L-MIND using patient-level data to isolate the tafasitamab contribution to the efficacy of the tafasitamab + LEN combination. Design: RE-MIND ( NCT04150328 ), a retrospective observational study, used a Nearest Neighbor ePS-based 1:1 Matching methodology to balance the LEN monotherapy cohort (observational data) and combination cohort (derived from L-MIND) for nine prespecified baseline covariates: age, Ann Arbor stage, last therapy refractoriness, number of prior therapy lines, primary refractoriness, prior ASCT, LDH, neutropenia, and anemia. The primary analysis set included matched patients who received a LEN starting dose of 25 mg/day (as in L-MIND) with sufficient follow-up. Patients: LEN monotherapy-treated patients from the US and EU who met the L-MIND-aligned eligibility criteria: adult patients with R/R DLBCL; 1–3 prior systemic therapies, including 1 CD20-targeting regimen; ASCT-ineligible. Baseline characteristics, LEN dosing, and patient outcomes were collected retrospectively from health records. Main outcome measures: The primary endpoint was investigator-assessed best objective response rate (ORR). Secondary endpoints included overall survival (OS) and complete response (CR) rates. Results: 490 patients were enrolled, of which 140 fulfilled the ePS matching criteria. Following matching, the primary analysis set included 76 patients from each cohort. Baseline characteristics were comparable. The primary endpoint was met. Best ORR was significantly higher in the combination cohort (67.1%) versus the monotherapy cohort (34.2%) (odds ratio 3.89; 95% confidence interval [CI]: 1.90–8.14; p Conclusions: Significantly better ORR, CR, and OS indicate potential synergistic effects of the tafasitamab + LEN combination in ASCT-ineligible R/R DLBCL. RE-MIND demonstrates the utility of real-world data in interpreting non-randomized trials.

Abstract 3320: Humanized NSG-Tg(Hu-IL15) mice support preclinical immune-oncology efficacy for testing of NK cells based immunology

Abstract The JAX® Onco-Hu® platform utilizes humanized mice engrafted with tumors to enable in vivo investigation of the interactions between the human immune system and human cancer. A major avenue of our investigation is to generate humanized mouse models containing a more complete human hematopoietic system and robust innate immune cell population. Nature killer (NK) cells are important players of innate defense against cancerous cells by a number of mechanisms, including antibody-dependent cell mediated cytotoxicity (ADCC). To overcome limited NK cell development in humanized NSG™ mice, we have developed NSG-Tg(Hu-IL15) mice constitutively expressing human IL15 in a physiological level. Following CD34+ HSC-engraftment of NSG-Tg(Hu-IL15) mice, significantly higher levels of human NK cells are detectable in peripheral blood as compared to NSG mice and the engraftment is stable for over six months. Levels of circulating human CD45+ cells, CD3+ T cells, and CD33+ myeloid cells are similar between the HSC-engrafted NSG-Tg(Hu-IL15) and NSG mice. Minimal baseline activation of NK cells is detected in the spleen and peripheral blood. Previously we have described that the human NK cells developing in HSC-engrafted NSG-Tg(Hu-IL15) mice are functional and can lyse the NK-sensitive target cells K562 by in vitro cytotoxicity assay. We also showed that NK cells directly limited growth of a PDX melanoma xenograft and NK cell depletion by antibody blockage largely abrogated the delayed PDX growth. Here we extended these observations by evaluating tumor growth of multiple PDX and CDX models in humanized NSG-Tg(Hu-IL15) mice. We found that not all tumors were able to grow and for those that grew some spontaneous regression might still occur. In ex vivo experiments NK cells in the PBMCs isolated from humanized NSG-Tg(Hu-IL15) mice could be activated by Her2+ human cancer cells in the presence of anti-human H2 antibody (Trastuzumab). Furthermore we showed in vivo that humanized NSG-Tg(Hu-IL15) mice mediated efficient ADCC against Daudi B lymphoma cells using anti-human CD20 antibody (Rituximab). Administration of rituximab resulted in significant tumor growth inhibition as measured by both tumor volume and tumor weight. Together these data demonstrate that HSC-engrafted NSG-Tg(Hu-IL15) mice support enhanced development of functional human NK cells and that this mouse model enables preclinical evaluation of NK cell-targeted cancer immunotherapy in vivo. Citation Format: Li-Chin Yao, Mingshan Cheng, Danying Cai, Leonard D. Shultz, James G. Keck. Humanized NSG-Tg(Hu-IL15) mice support preclinical immune-oncology efficacy for testing of NK cells based immunology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3320.

Abstract LB-227: Preclinical development and characterization of STI-6129, an anti-CD38 antibody-drug conjugate, as a new therapeutic agent for multiple myeloma

Abstract CD38 is a validated target for the treatment of CD38-overexpressing hematologic malignancies such as multiple myeloma (MM). Daratumumab is the first anti-CD38 mAb drug approved by the FDA for use as single agent and in combination with standard therapies for MM. However, resistance to Daratumumab in both primary and refractory MM patients has been reported. Here we report the preclinical development of STI-6129 (also CD38-077), a CD38-targeting antibody-drug conjugate (ADC), as a new therapeutic agent for MM. STI-6129 consists of a fully human anti-CD38 antibody, STI-5171, identified from the Sorrento G-MAB® antibody library, conjugated to the microtubule inhibitor duostatin-5.2 (Duo-5.2) via a non-polyethylene glycol linker using our site-specific conjugation proprietary C-LOCK technology. STI-5171 binds specifically to CD38-positive but not CD38-negative tumor cell lines with a KD ~ 1.72E-8 M. STI-6129 is internalized into CD38-positive cells at a rate comparable to that of the unconjugated antibody STI-5171. In cytotoxicity studies, STI-6129 exhibited CD38-dependent cytotoxic activity against a panel of CD38-expressing tumor cell lines with the range of EC50 from 5-100 µg/mL. Importantly, STI-6129 does not display cytotoxic activity (EC50 > 150 μg/mL) against normal human PBMCs after 120 hr exposure. Mechanistic evaluation of STI-5171 compared to Daratumumab revealed that this antibody does not induce homotypic aggregation of tumor cells, a cell adhesion response that has been reported to be linked to drug resistance and increased metastatic potential. Further, the anti-tumor activity of STI-6129 showed broad, potent, and CD38-dependent in vivo efficacy in multiple xenograft animal models. Pharmacokinetic evaluation of STI-6129 in Daudi-Fluc tumor-bearing mice revealed that the ADC is stable with half-life (T1/2) of 7-11 days, comparable to that of the unconjugated antibody. The serum concentration of STI-6129 remained above the therapeutically effective level up to 1 week in mice after a single injection at 10 mg/kg. In cynomolgus monkey, the PK profiles of STI-6129 and STI-5171 were nearly identical, further confirming the stability of the ADC in monkey blood circulation.In summary, STI-6129 exhibits potent in vitro and in vivo anti-tumor activities in multiple CD38-positive hematological models. These results warrant further development of STI-6129, potentially as a better or alternative agent for treatment of multiple myeloma. Citation Format: Lingna Li, Andrew Hau, Wenyong Tong, Megan Lau, Tong Zhu, Katherine Fells, Angel Wei, Xiaoqing Li, Dylan Deng, Yingqing Sun, Ernest Kovacs, Alisher Khasanov, Zheng Yan, Panyun Zhu, Heyue Zhou, Henry Ji, Hui Li, Hong Zhang. Preclinical development and characterization of STI-6129, an anti-CD38 antibody-drug conjugate, as a new therapeutic agent for multiple myeloma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-227.

Immunohistochemical identification of T and B lymphocytes in formalin-fixed paraffin-embedded chicken tissues using commercial antibodies

Immunohistochemical method to detect avian lymphocytes is an efficient and reliable tool for accurate diagnosis, and immunological analysis of avian diseases. However, there are scarce studies reporting immunohistochemistry (IHC) using commercially available antibodies against lymphocytes in formalin-fixed paraffin-embedded (FFPE) chicken tissues. In the present study, we established an immunohistochemical method to identify chicken T and B lymphocytes in FFPE chicken tissues using commercial antibodies against chicken or human antigens. For this IHC method, the five tested anti-T lymphocyte antibodies reacted with chicken T lymphocytes on the FFPE sections. Further, 10 commercial anti-B lymphocyte antibodies were tested; of these, three successfully detected chicken B lymphocytes for IHC. In particular, anti-human CD3 (clone F7.2.38) antibody was most suitable for the detection of chicken T lymphocytes, whereas anti-chicken B cell activating factor receptor (BAFF-R) antibody (clone 2C4) was most suitable for the detection of chicken B lymphocytes under our IHC staining conditions. These two antibodies reacted with numerous lymphocytes of all representative lymphoid tissues without problematic background staining and nonspecific reactions. Our results indicate that T and B lymphocytes in FFPE chicken tissues can be immunohistochemically detected using commercial antibodies.

Gemtuzumab Ozogamicin Combined With Intensive Chemotherapy in Patients With Acute Myeloid Leukemia Relapsing After Allogenic Stem Cell Transplantation

BackgroundMore than one-third of patients with acute myeloid leukemia (AML) will relapse after allogenic hematopoietic cell transplant (allo-HCT). The main challenge is to overcome disease resistance to achieve a new complete remission while avoiding excessive toxicity. Gemtuzumab ozogamicin (GO), a conjugate of calicheamicin linked to the humanized monoclonal anti-CD33 antibody, has been used for refractory or relapsed AML with promising response rates, but liver toxicity of GO has long been considered a limiting factor. Patients and MethodsWe included 18 consecutive patients with AML relapsing after a first allo-HCT and treated with fractioned GO (fGO) and intensive chemotherapy. The median age was 40 years (range, 18-65). ResultsThe overall response rate was 72% (13/18), including 7 complete remissions. No death was attributed to treatment toxicity. The main liver toxicity was transient and consisted of transaminase level elevation and hyperbilirubinemia. No cases of veno-occlusive disease were observed after the GO treatment. From the time of salvage treatment initiation, 1- and 2-year OS rates were 54% (95% confidence interval, 28%-74%) and 42% (95% confidence interval, 19%-63%), respectively. ConclusionsOur study suggests the feasibility, efficacy, and safety of an fGO-based salvage regimen combined with intensive chemotherapy in patients with CD33+ AML in the case of early relapse after an allo-HCT.

Coreceptor blockade targeting CD4 and CD8 allows acceptance of allogeneic human pluripotent stem cell grafts in humanized mice

We have previously demonstrated that short-term coreceptor blockade with non-lytic monoclonal antibodies enables the long-term survival of fully allogeneic embryonic stem cell (ESC) transplants in mice. Here, we describe the use of Hu-PBL humanized mice to determine whether short-term coreceptor blockade with humanized anti-human CD4 and CD8 antibodies can achieve the same outcome towards human ESC derivatives. While control Hu-PBL mice rejected allogeneic hESC-derived transplants within weeks, mice treated with coreceptor blocking antibodies held their grafts for 7 weeks, the duration of the study. Rejection in the control mice was associated with demonstrable infiltrates of human CD45 white blood cells, predominantly of CD8 T-cells, whereas anti-CD4, but not anti-CD8 antibody treated mice showed remarkably reduced lymphocyte infiltration and prolonged allograft survival, indicating that the CD4+ T-cells were crucial to the rejection process. Our results give support to the principle that short-term blockade of T-cell co-receptors can achieve long-term acceptance of regenerative cell transplants in humans.

Abstract B72: Humanized NSG-Tg(Hu-IL15) mice support preclinical immune-oncology efficacy for testing of NK cell-based immunotherapy

Abstract The JAX® Onco-Hu® platform utilizes humanized mice engrafted with tumors to enable in vivo investigation of the interactions between the human immune system and human cancer. We have shown that humanized NOD-scid IL2Rγ null (NSG) mice bearing patient-derived xenografts (PDX) allow efficacy studies of checkpoint inhibitors. A major avenue of our investigation is to generate humanized mouse models containing a more complete human hematopoietic system and robust innate immune cell population. Nature killer (NK) cells are important players of innate defense against cancerous cells by a number of mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC). To overcome limited NK cell development in humanized NSG mice, we have developed NSG-Tg(Hu-IL15) (JAX stock number 030890) mice constitutively expressing human IL15. Following HSC-engraftment of NSG-Tg(Hu-IL15) mice, significantly higher levels of human CD16+CD56+ NK cells are detectable as compared to NSG mice in peripheral blood during the whole course of the study (18 weeks). Levels of circulating human CD45+ cells, CD3+ T cells, CD19+ B cells, and CD33+ myeloid cells are similar between the HSC-engrafted NSG-Tg(Hu-IL15) and NSG mice. Previously we have described that the human NK cells developing in HSC-engrafted NSG-Tg(Hu-IL15) mice are functional and can lyse the NK-sensitive target cells K562 by in vitro cytotoxicity assay. We also showed that NK cells limit the growth of a PDX melanoma xenograft. Here we show that humanized NSG-Tg(Hu-IL15) mice mediate efficient ADCC against Daudi B lymphoma cells in vivo. Administration of anti-human CD20 antibody (rituximab) resulted in significant tumor growth inhibition, in both tumor volume and tumor weight. We observed consistent ADCC efficacy in humanized NSG-Tg (Hu-IL15) mice engrafted with three different HSC donors. Together these data demonstrate that HSC-engrafted NSG-Tg(Hu-IL15) mice support enhanced development of functional human NK cells and that this mouse model enables NK cell-targeted cancer immunotherapy for preclinical testing. Citation Format: Li-Chin Yao, Mingshan Cheng, Leonard D. Shultz, Dale L. Greiner, Michael A. Brehm, James G. Keck. Humanized NSG-Tg(Hu-IL15) mice support preclinical immune-oncology efficacy for testing of NK cell-based immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B72.

Development of AO-176, a Next-Generation Humanized Anti-CD47 Antibody with Novel Anticancer Properties and Negligible Red Blood Cell Binding.

Inhibitors of adaptive immune checkpoints have shown promise as cancer treatments. CD47 is an innate immune checkpoint receptor broadly expressed on normal tissues and overexpressed on many tumors. Binding of tumor CD47 to signal regulatory protein alpha (SIRPα) on macrophages and dendritic cells triggers a "don't eat me" signal that inhibits phagocytosis enabling escape of innate immune surveillance. Blocking CD47/SIRPα interaction promotes phagocytosis reducing tumor burden in numerous xenograft and syngeneic animal models. We have developed a next-generation humanized anti-CD47 antibody, AO-176, that not only blocks the CD47/SIRPα interaction to induce tumor cell phagocytosis, but also induces tumor cytotoxicity in hematologic and solid human tumor cell lines, but not normal noncancerous cells, by a cell autonomous mechanism (not ADCC). AO-176 also binds preferentially to tumor versus many normal cell types. In particular, AO-176 binds negligibly to RBCs in contrast to tumor cells, even at high concentrations up to 200 μg/mL and does not agglutinate RBCs up to 1 mg/mL in vitro These properties are expected not only to decrease the antigen sink, but also to minimize on-target clinical adverse effects observed following treatment with other reported RBC-binding anti-CD47 antibodies. When tested in cynomolgus monkeys, AO-176 was well tolerated with no adverse effects. Finally, we show that AO-176 demonstrates dose-dependent antitumor activity in tumor xenograft models. Taken together, the unique properties and antitumor activity of our next-generation anti-CD47 antibody, AO-176, distinguishes it from other CD47/SIRPα axis targeting agents in clinical development.