CD3 mAb Research Articles

PKC agonism restricts innate immune suppression, promotes antigen cross-presentation and synergizes with agonistic CD40 antibody therapy to activate CD8+ T cells in breast cancer

Myeloid-derived suppressor cells (MDSCs) are an immature innate cell population that expands in pathological conditions such as cancer and suppresses T cells via production of immunosuppressive factors. Conversely, efficient cytotoxic T cell priming is dependent on the ability of antigen-presenting cells (APCs) to cross-present tumor antigens to CD8+ T cells, a process that requires a specific subtype of dendritic cells (DCs) called conventional DC1 (cDC1) which are often dysfunctional in cancer. One way to activate cDC1 is ligation of CD40 which is abundantly expressed by myeloid cells and its agonism leads to myeloid cell activation. Thus, targeting MDSCs while simultaneously expanding cross-presenting DCs represents a promising strategy that, when combined with agonistic CD40, may result in long-lasting protective immunity. In this study, we investigated the effect of PKC agonists PEP005 and prostratin on MDSC expansion, differentiation, and recruitment to the tumor microenvironment. Our findings demonstrate that PKC agonists decreased MDSC expansion from hematopoietic progenitors and induced M-MDSC differentiation to an APC-like phenotype that expresses cDC1-related markers via activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Simultaneously, PKC agonists favored cDC1 expansion at the expense of cDC2 and plasmacytoid DCs (pDC). Functionally, PKC agonists blunted MDSC suppressive activity and enhanced MDSC cross-priming capacity both in vitro and in vivo. Finally, combination of PKC agonism with agonistic CD40 mAb resulted in a marked reduction in tumor growth with a significant increase in intratumoral activated CD8+ T cells and tissue-resident memory CD8+ T cells in a syngeneic breast cancer mouse model. In sum, this work proposes a novel promising strategy to simultaneously target MDSCs and promote APC function that may have highly impactful clinical relevance in cancer patients.

Regulation of CD38 on Multiple Myeloma and NK Cells by Monoclonal Antibodies.

CD38 is highly expressed on multiple myeloma (MM) cells and plays a role in regulating tumor generation and development. CD38 monoclonal antibodies (mAbs) have been used as an effective therapy for MM treatment by various mechanisms, including complement-dependent cytotoxic effects, antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, programmed cell death, enzymatic modulation, and immunomodulation. Although CD38 mAbs inhibit the proliferation and survival of MM cells, there are substantial side effects on antitumoral NK cells. The NK-mediated immune response needs to be further evaluated to minimize the adverse effects of NK cell loss. The killing effect of CD38 mAbs on CD38high NK cells should be minimized and the potential combination of CD38low/- NK cells and CD38 mAbs should be maximized to better benefit from their therapeutic efficacy against MM. CD38 mAb effects against MM can be maximized by combination therapies with immunomodulatory imide drugs (IMiDs), proteasome inhibitors (PIs), anti-programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) antibodies, or cellular therapies for the treatment of MM, especially in patients with relapsed or refractory MM (R/R MM) and drug-resistant MM.

Dual-targeting vaccine of FGL1/CAIX exhibits potent anti-tumor activity by activating DC-mediated multi-functional CD8 T cell immunity

Tumor DNA vaccine as an effective therapeutic approach can induce systemic immunity against malignant tumors, but its therapeutic effect is still not satisfactory in advanced renal cancer. Herein, a novel DNA vaccine containing dual antigens of fibrinogen-like protein 1 (FGL1) and carbonic anhydrase IX (CAIX) was developed and intramuscularly delivered by PLGA/PEI nanoparticles for renal cancer therapy. Compared with PLGA/PEI-pCAIX immunization, PLGA/PEI-pFGL1/pCAIX co-immunization significantly inhibited the subcutaneous tumor growth and promoted the differentiation and maturation of CD11c+ DCs and CD11c+CD11b+ DCs subset. Likewise, the increased capabilities of CD8 T cell proliferation, CTL responses, and multi-functional CD8+ T cell immune responses were observed in PLGA/PEI-pFGL1/pCAIX vaccine group. Interestingly, depletion of CD8+ T cells by using CD8 mAb resulted in a loss of anti-tumor function of PLGA/PEI-pFGL1/pCAIX vaccine, suggesting that the anti-tumor activity of the vaccine was dependent on CD8+ T cell immune responses. Furthermore, PLGA/PEI-pFGL1/pCAIX co-immunization also suppressed the lung metastasis of tumor mice by enhancing the multi-functional CD8+ T cell responses. Therefore, these results indicate that PLGA/PEI-pFGL1/pCAIX vaccine could provide an effective protective effect for renal cancer by enhanced DC-mediated multi-functional CD8+ T cell immune responses. This vaccine strategy offers a potential approach for solid or metastatic tumor treatment.

Impact of IFN-y and CD40 signalling on Toxoplasma gondii cyst formation in differentiated Neuro-2a neuroblastoma cells.

Signalling by IFN-y and CD40 is known to trigger anti-microbial activity in macrophages infected with Toxoplasma gondii, but their effects on infected neurons are less well known. Here, we compared how stimulation with IFN-y and an agonistic anti-CD40 mAb impacts infection and cyst formation in the mouse neuroblastoma cell line Neuro-2a relative to bone marrow-derived macrophages. Both IFN-y and CD40 mAb decreased cyst emergence in Neuro-2a cells. In macrophages, these stimuli decreased infection, but had no impact on infection in the neuroblastoma cell line. Resistance to killing in Neuro-2a cells may explain why neurons preferentially harbour parasites during chronic infection in the brain.

Immunotherapy for Tumor Metastasis by Artificial Antigen-Presenting Cells via Targeted Microenvironment Regulation and T-Cell Activation.

Effective expansion of T-cells without ex vivo stimulation and maintenance of their antitumor functions in the complex tumor microenvironment (TME) are still daunting challenges in T-cell-based immunotherapy. Here, we developed biomimetic artificial antigen-presenting cells (aAPCs), ultrathin MnOx nanoparticles (NPs) functionalized with T-cell activators (anti-CD3/CD28 mAbs, CD), and tumor cell membranes (CMs) for enhanced lung metastasis immunotherapy. The aAPCs, termed CD-MnOx@CM, not only efficiently enhanced the expansion and activation of intratumoral CD8+ cytotoxic T-cells and dendritic cells after homing to homotypic metastatic tumors but also regulated the TME to facilitate T-cell survival through catalyzing the decomposition of intratumoral H2O2 into O2. Consequently, the aAPCs significantly inhibited the development of lung metastatic nodules and extended the survival of a B16-F10 melanoma metastasis model, while minimizing adverse events. Our work represents a new biomaterial strategy of inhibiting tumor metastasis through targeted TME regulation and in situ T-cell-based immunotherapy.

Immunosuppressive activity of a cycloartane triterpene glycoside from Beesia calthaefolia by inhibiting T cell proliferation

BC-1 is a cycloartane triterpene glycoside isolated from the whole plant of Beesia calthaefolia. Our recent studies proved that BC-1 inhibited proliferation of splenic lymphocyte and phagocytosis of macrophages, and inhibited the increased production of TNF-α and IL-1β. However, it lacks of study about the immunomodulatory effect of BC-1 on purified T lymphocytes. Therefore, in the present study, we evaluated the suppressive potentials of BC-1 on immune responses in vitro. BC-1 markedly suppressed anti-CD3/CD28 mAbs (mAbs) induced murine T lymphocytes proliferation, the expression levels of CD69 and CD25 of CD3+ T cells. BC-1 could strongly decrease ratio of CD4+/CD8+, decrease the Th1/Th2 cytokines production (IL-2, IFN-γ, IL-4, and IL-10) of CD4+ T-cells. In addition, we studied signal transduction pathways about T-cell activation on puried murine CD4+ T lymphocytes by western-blot assay. The data revealed that BC-1 could inhibit the activation of JNK, ERK and PI3K/AKT signal transduction pathways. These results indicated that BC-1 possesses potential downregulating effect on the immune system and might be developed as an immunosuppressive agent in treatment of CD4+ T cell-mediated inflammatory and undesired immune responses.

Retrospective Single-Institution Analysis of Patients with Chronic Lymphocytic Leukemia with TP53 alterations Treated First-Line with Bruton's Tyrosine Kinase Inhibitor-Based Therapy

Retrospective Single-Institution Analysis of Patients with Chronic Lymphocytic Leukemia with TP53 alterations Treated First-Line with Bruton's Tyrosine Kinase Inhibitor-Based Therapy

Real-World Evaluation of the Treatment Landscape for Chronic Lymphocytic Leukemia

Introduction:The treatment paradigm of chronic lymphocytic leukemia (CLL) has changed significantly with the introduction of targeted oral therapies, including Bruton's tyrosine kinase inhibitors (BTKis) and B-cell lymphoma 2 inhibitors (Bcl-2is). As more therapies enter the clinic, real-world data (RWD) are important to understand changes in the CLL treatment landscape and describe treatment patterns, to guide decisions on treatment strategies.Methods:This retrospective analysis used 2 RWD sources. Patients (pts) diagnosed with CLL and treated with chemotherapy, CD20 monoclonal antibodies (mAbs), BTKis, or Bcl-2is were studied in the Optum Clinformatics® Data Mart (CDM), a de-identified US administrative claims database. Pts in the CDM analysis received at least 1 prescription for or administration of a treatment of interest between 2016 and 2020 Q2, and a diagnosis of CLL within 12 months (mo) of treatment start. Cross-sectional annual cohorts were used to understand changes in treatment mix over time, and longitudinal cohorts were formed to identify treatment patterns. Medication adherence was assessed with persistence at 12 mo using a 60-day gap and proportion of days covered (PDC) at 12 mo. Electronic health records from oncology practices were extracted through December 2020 from the COTA real-world evidence database. Adults diagnosed with CLL/small lymphocytic lymphoma and treated with BTKis and/or Bcl-2is were included. Treatment discontinuation was assessed using physician-documented treatment dates and reasons for discontinuation, and overall duration of treatment with targeted therapy was assessed using Kaplan-Meier analysis.Results:In CDM, annual treatment rate among pts diagnosed with CLL increased from 16% (1788/10911) in 2016 to 26% (2441/9349) in 2020. The proportion of pts treated with BTKis and Bcl-2is increased from 40% (2016) to 65% (2020) and <1% (2016) to 13% (2020), respectively; corresponding decreases in the use of chemotherapies and CD20 mAbs were observed (Figure 1A). In pts initiating BTKis, most pts were not previously treated (937/1438; 65%); the most common initial regimens were BTKi monotherapy (mono) (1367/1438; 95%) and BTKi + CD20 mAb (54/1438; 4%). Few pts switched from BTKi mono to CD20 mAb mono (43/1367; 3%) or added CD20 mAb to BTKi mono (44/1367; 3%) within 12 mo. In pts initiating Bcl-2is, most received prior treatment (169/198; 85%); most common regimens were Bcl-2i mono (159/198; 80%), Bcl-2i + CD20 mAb (31/198; 16%), and Bcl-2i + BTKi (8/198; 4%). Among pts initiating Bcl-2i mono, 30% (48/159) added CD20 mAb within 12 mo; among pts initiating Bcl-2i + CD20 mAb, 94% (29/31) switched to Bcl-2i mono within 12 mo. Persistence at 12 mo was similar for BTKis (899/1438; 63%) and Bcl-2is (120/198; 61%) and mean (SD) PDC was similar for BTKis 0.69 (0.31) and Bcl-2is 0.68 (0.29). In COTA, 469 pts treated with BTKis and/orBcl-2is were included. More pts initiated BTKis as first-line (1L) (185/449; 41%) compared with Bcl-2is (13/109; 12%). Among pts initiating targeted therapy as 1L, most pts (81%; 149/185) initiated BTKi mono. Discontinuation rates were high for pts receiving BTKis (62%; 279/449) and Bcl-2is (61%; 67/109). Among BTKi discontinuers, toxicity (54%; 150/279) was the most common reason for discontinuation, followed by disease progression (12%; 34/279) (Figure 1B). Among Bcl-2i discontinuers, toxicity (36%; 24/67) was the most common reason for discontinuation, followed by completion of regimen (30%; 20/67) (Figure 1C). Median duration of treatment was 777 days (95% CI: 632-920) for the BTKis group and 639 days (95% CI: 335-755) for the Bcl-2is group. Median treatment duration in days was comparable between mono and combination regimens in both BTKis (782 vs. 809) and Bcl-2is (747 vs. 718) groups.Conclusions:RWD show standard of care shifted from chemoimmunotherapy to targeted therapies. Pts receiving targeted therapies had high rates of discontinuation, most commonly due to toxicity. Most targeted regimens were mono; more BTKi pts were treatment naive compared to Bcl-2i pts. Slow adoption of fixed-duration therapy with the Bcl-2i combination regimen was observed. The duration of Bcl-2i mono was comparable to that of the Bcl-2i combination regimens, and a high number of pts initiating combo regimens continued on mono regimens in contrast to regimens studied in randomized controlled trials. [Display omitted] DisclosuresSmith: Novartis: Current Employment; Pfizer: Ended employment in the past 24 months. Owusu: Novartis: Current Employment; Roche: Ended employment in the past 24 months. Wormser: Roche: Current equity holder in publicly-traded company; Novartis: Current Employment, Current equity holder in publicly-traded company. Woo: Novartis: Current Employment, Current equity holder in publicly-traded company.

IPSC-Derived Natural Killer Cells and Macrophages Synergistically Kill Acute Myeloid Leukemia Blasts

Despite many advances, the treatment of acute myeloid leukemia (AML) remains challenging, and few patients are cured by therapies other than allogeneic hematopoietic cell transplant. Treatment with natural killer (NK) cells from allogeneic donors is a promising therapy that can achieve remissions in 30-50% of AML patients. To generate an improved cell-based therapy for AML, our group has produced NK cells from induced pluripotent stem cells (iPSCs). iPSC-derived NK cells effectively kill AML cells, but may benefit from additional modifications or combination with other therapies to durably cure AML. Based on studies that demonstrate that targeting the CD47 pathway on macrophages and NK cells improves anti-tumor activity and is an effective treatment for patients with AML, we investigated the combination of iPSC-derived NK cells with iPSC-derived macrophages with and without CD47 blockade for the treatment of AML.AML clinical trials combining anti-CD47 monoclonal antibodies (mAb) with chemotherapy have demonstrated an anti-tumor effect primarily thought to be mediated though a macrophage immune-checkpoint blockade mechanism. To determine if addition of iPSC-derived macrophages can improve the cytotoxicity of NK cells against AML blasts, we co-cultured iPSC-NK cells and MOLM13 or MV-4-11 AML cells with iPSC-macrophages in a standard cytotoxicity assay. Similar results were found for cytotoxicity tests against both AML cell lines (Figure panels A and B). While macrophages alone did not kill AML blasts, the addition of iPSC-macrophages to iPSC-NK cells significantly improved killing of the AML cells by 50% (p<0.01). Addition of an anti-CD47 mAb (B6H12) further increased killing of these AML cells by the iPSC-NK cell + iPSC-macrophage combination treatment by an additional 23% (p<0.01). Intriguingly, the addition of just the anti-CD47 mAb to the iPSC-NK cells also significantly increased anti-AML activity, although this increased killing was consistently lower than what was seen with addition of iPSC-macrophages combined with anti-CD47 and iPSC-NK cells. Addition of the CD47 mAb to iPSC-macrophages without NK cells did not result in increased anti-AML cytotoxicity. We also demonstrated that blockade of SIRPα (the receptor on NK cells for CD47) significantly increased NK cell killing of AML blasts by 16% (p<0.05). Furthermore, the combination of iPSC-NK cells + iPSC-macrophages + SIRPα mAb led to a 37% increase in cytotoxicity compared to iPSC-NK cells + iPSC-macrophages alone (p<0.01).To confirm that addition of anti-CD47 or anti-SIRPα antibodies increased NK cell activation via loss of the inhibitory CD47-SIRPα interaction between NK cells and AML blasts and not by another mechanism, we tested the effect of adding both CD47 mAb and anti-SIRPα antibodies. Compared to addition of either anti-CD47 or anti-SIRPα mAb alone, the combination induced no additional increase in anti-AML activity by the NK cells. These results suggest that the CD47-SIRPα interaction between AML and NK cells is an important inhibitory immune-checkpoint on NK cells. To control for the effect of blocking SIRPα on macrophages, we tested the addition of the SIRPα mAb to iPSC-macrophages. This combination did not improve on the lack of cytotoxicity exhibited by macrophages alone. We also evaluated if ADCC mediated by the anti-CD47 mAb binding AML blasts could account for the increase in cytotoxicity and found that blockade of Fc-receptors on NK cells does not diminish the increase in cytotoxicity seen with addition of the mAb, excluding a role for ADCC.To investigate if these findings with AML could be extended to other hematologic malignancies, we tested the combination of iPSC-NK cells, iPSC-macrophages and CD47 mAb against the RPMI-8226 multiple myeloma cell line (Figure C). Here, we again demonstrate that CD47 blockade combined with iPSC-macrophages leads to increased NK cell-mediated anti-myeloma activity. In vivo studies testing the combination of iPSC-NK cells, iPSC-macrophages and CD47 mAb against human AML and myeloma cells in mouse xenograft-models are ongoing. Together our results indicate that blocking the CD47-SIRPα interaction between NK cells and tumor cells consistently mediates improved anti-tumor activity. Furthermore, iPSC-derived NK cells and macrophages provide an important, standardized, “off-the-shelf” cell therapy approach that can be translated into novel clinical therapies. [Display omitted] DisclosuresKaufman: Shoreline Biosciences: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qihan Biotech: Consultancy, Current holder of stock options in a privately-held company; VisiCELL Medical: Consultancy, Current holder of stock options in a privately-held company.

Venetoclax, Obinutuzumab and Atezolizumab (PD-L1 Checkpoint Inhibitor) for Treatment for Patients with Richter Transformation

Venetoclax, Obinutuzumab and Atezolizumab (PD-L1 Checkpoint Inhibitor) for Treatment for Patients with Richter Transformation

Venetoclax, Obinutuzumab and Atezolizumab (PD-L1 Checkpoint Inhibitor) for First-Line Treatment for Patients with Chronic Lymphocytic Leukemia (CLL)

Venetoclax, Obinutuzumab and Atezolizumab (PD-L1 Checkpoint Inhibitor) for First-Line Treatment for Patients with Chronic Lymphocytic Leukemia (CLL)

A Novel B Cell Antigen Designated Lambda Myeloma Antigen (LMA) Has Been Identified Using Two Fully Human Monoclonal Antibodies (Mabs) That Bind to Similar Epitopes on Plasma Cells from Patients with Plasma Cell Dyscrasias

Introduction:The novel kappa (κ) myeloma antigen (KMA) has been described and a specific monoclonal antibody KappaMab (formerly MDX-1097) developed which is currently in a Phase IIb clinical trial. In this study 2 human LambdaMabs (10B3 and 7F11) were shown to specifically bind to a conformational epitope in the lambda (λ) light chain constant region when it is held in non-covalent association with lipids in the cell membrane (LMA). The 10B3 Mab binds to all λ isotypes and 7F11 binds to isotypes 2 and 3; neither antibody binds κ light chain or immunoglobulin (Igλ or Igκ).Methods:To detect LMA on λ+ myeloma cell lines and on λ-restricted patient bone marrow (BM) samples, 7F11 and 10B3 Fab'2 fragments were conjugated to APC and R-PE and used to stain the λ myeloma cell lines LP-1, RPMI8226 and OPM-2 followed by flow cytometry analysis. KappaMab Fab'2 (KMA Fab'2) and the κ myeloma cell line JJN3 were used as a negative control and to identify KMA on κ-restricted patient BM samples.Patient BM samples (κ=43 and λ=22) included Monoclonal Gammopathy of Undetermined Significance (MGUS), untreated and treated multiple myeloma (MM) patients, AL amyloidosis, plasmacytoma and Waldenstroms macroglobulinemia (WM). Multiparametric FCM immunophenotyping was performed with the 10B3 Fab'2 fragments and CD38, CD138, CD269 (BCMA), CD319 (SLAM F7), CD56 and CD45 Mabs. Plasma cells (PCs) were identified by the co-expression of CD38 and CD138, then CD138+/CD38+ gated cells were analyzed for 10B3 or KMA, CD269, CD319, and CD56. KMA and LMA Fab'2 fragments were used as negative controls for each other. The antigen density of KMA or LMA versus BCMA on PCs in 55 samples was assessed using Quantibrite beads.Immunohistochemistry (ICH) tissue cross-reactivity studies using validated automated methods on tissue with whole antibody 7F11-biotin and 10B3-FITC were performed on MM cell lines, λ myeloma lung tissue (plasmacytoma) and a panel of 38 normal human tissues. Serial sections from snap frozen blocks were used to retain the conformational epitope and then stained with the λ Mabs and visualised by light microscopy.Results:The conjugated 10B3 Fab'2 fragment bound to LMA-expressing cell lines LP-1, RPMI8226 and OPM-2 (isotypes 1-3) and 7F11 bound to RPMI8226 and OPM-2, (isotypes 2-3); neither bound JJN3. KMA Fab'2 did not bind to λ myeloma cell lines but bound JJN3.Expression profiles for patient BM samples (Table 1) showed KMA was expressed on PCs from untreated (N=9/17; 53%) and treated (N=8/11; 73%) MM samples, whereas BCMA expression was 88% (N=15/17) and 82% (N=9/11) respectively. BM PCs from all 3 plasmacytoma cases and 1 WM case were positive for both KMA and BCMA. BM PCs from MGUS cases were all positive for BCMA and positive for KMA in half the cases studied. The expression of KMA and CD56 was highest on PCs from treated MM patient samples. Antigen density for KMA and BCMA was similar in the untreated patients. In treated patients KMA density was higher than BCMA, other samples had lower antigen density of both KMA and BCMA.LMA (Table 2) and BCMA were expressed on 50% and 90% of untreated MM samples and all LMA+ samples co-expressed BCMA but only 1 co-expressed CD56. All treated λMM samples expressed BCMA and 60% expressed LMA. LMA was positive on PCs from the 3 amyloidosis samples, BCMA was expressed weakly in only 1 of these whereas CD56 was always co-expressed with LMA. MGUS and WM samples did not express LMA. Similar to KMA, the antigen density of LMA and BCMA was equivalent in untreated patients but in treated patients LMA density was higher than BCMA. All 3 amyloidosis samples were λ isotype.IHC results showed that 10B3 and 7F11 bound to myeloma cell lines and 10B3 bound to PCs in λ plasmacytoma sections. Both Mabs bound occasional PCs or dendritic cells in the GI tract mucosa, tonsil and various secondary lymphoid organs. No off-target binding of 10B3 and 7F11 was observed and both antibodies bound occasional PCs in secondary lymphoid tissue.Conclusion:These studies used mostly myeloma samples and a small number of other plasma cell dyscrasias. Nevertheless expression of KMA and LMA was identified on PCs across the spectrum of disease. Within the treated patient cohort the antigen density of KMA or LMA was higher than that of BCMA and implies there is an enrichment of these novel antigens in relapsed refractory myeloma. No off-target binding was observed in normal human tissues and binding was limited to occasional leukocytes in secondary lymphoid tissue. [Display omitted] DisclosuresHu: HaemaLogiX Pty Ltd: Current Employment. Dunn: HaemaLogiX Pty Ltd: Current Employment.

Clinical Trials of BCMA-Targeted CAR-T Cells Utilizing a Novel Non-Viral Transposon System

Clinical Trials of BCMA-Targeted CAR-T Cells Utilizing a Novel Non-Viral Transposon System

P-226: Clinical outcomes of Multiple Myeloma patients after anti-CD38 monoclonal antibodies failure

<h3>Background</h3> Anti-CD38 monoclonal antibodies (Mab) have significantly improved the prognosis of patients with multiple myeloma. However, not all patients sustain durable responses. Patients relapsed or refractory (R/R) to anti-CD38 Mab present dismal prognosis and constitute a group for whom therapeutic needs are still not defined. We aimed to describe the natural history of patients relapsed or refractory to CD38 monoclonal antibodies. <h3>Methods</h3> Unicentric retrospective analysis of 57 MM patients R/R to anti-CD38 Mab. Refractoriness was defined as progression within the first two months of treatment. We used chi-square test to compare categorical variables, the Kaplan-Meier method and log rank test for the survival analysis and multivariable analysis using logistic regression. IBM SPSS (v25.0) was used for the statistical analysis. <h3>Results</h3> The median age was 66 years, 57.9% were female. The cohort received a median of 2; R(0-11) lines of treatment previous to anti-CD38 Mab. 49% had undergone autologous transplant and 64.9% had received a proteasome inhibitor and an immunomodulatory previously. 21 patients (37%) were refractory and 20 (35%) achieved very good partial response or better response. The most frequent combinations were with lenalidomide (DRd; 11%), bortezomib (DVd; 16%), bortezomib and melphalan (D-VMP; 13%) and with carfilzomib (KDd; 27%). 26% received daratumumab in monotherapy or with corticosteroids. Median time to treatment progression was 7 months and median overall survival (OS) was 12 months for the global cohort. OS was longer in patients who achieved a deeper response (49, 12 and 4 months for patients with complete response, partial response and stable disease or progression respectively; p=0.001). High cytogenetic risk was related to poor outcomes, with an OS of 5 months and more frequent CD38 Mab refractoriness (63% vs 37% for the global cohort; p=0.007). Resistance to daratumumab as a first line treatment was observed in 8 patients. 7 of them were treated within a clinical trial for non eligible AHCT patients Median time to daratumumab progression within this group was 12 months and median OS was 49 months. 49 (86%) of the patients received at least one line of treatment after anti-CD38 Mab R/R and 47% of them had and objective response to at least one of the subsequent therapies employed, with median progression free survival (PFS) of 5 months. Daratumumab-resistant patients had longer OS when treated with regimens containing pomalidomide compared to other agents (median OS 26 vs 6 months respectively; p=0.021). <h3>Conclusion</h3> Patients R/R to CD38 targeted monoclonal antibodies present dismal prognosis, with a median OS of 12 months. Patients with high cytogenetic risk have worse outcomes, with shorter OS and higher probability to anti-CD38 Mab refractoriness. Treatment with pomalidomide-based regimens may be an interesting option for this population. The authors declare no conflicts of interest.

Converting an Anti-Mouse CD4 Monoclonal Antibody into an scFv Positron Emission Tomography Imaging Agent for Longitudinal Monitoring of CD4+ T Cells.

Immuno-positron emission tomography (PET), a noninvasive imaging modality, can provide a dynamic approach for longitudinal assessment of cell populations of interest. Transformation of mAbs into single-chain variable fragment (scFv)-based PET imaging agents would allow noninvasive tracking in vivo of a wide range of possible targets. We used sortase-mediated enzymatic labeling in combination with PEGylation to develop an anti-mouse CD4 scFv-based PET imaging agent constructed from an anti-mouse CD4 mAb. This anti-CD4 scFv can monitor the in vivo distribution of CD4+ T cells by immuno-PET. We tracked CD4+ and CD8+ T cells in wild-type mice, in immunodeficient recipients reconstituted with monoclonal populations of OT-II and OT-I T cells, and in a B16 melanoma model. Anti-CD4 and -CD8 immuno-PET showed that the persistence of both CD4+ and CD8+ T cells transferred into immunodeficient mice improved when recipients were immunized with OVA in CFA. In tumor-bearing animals, infiltration of both CD4+ and CD8+ T cells increased as the tumor grew. The approach described in this study should be readily applicable to convert clinically useful Abs into the corresponding scFv PET imaging agents.

CD40 Agonist Monoclonal Antibody-Mediated Hepatitis in TNF-Receptor 1 Gene Knockout Mice.

Tumor necrosis factor-alpha (TNF-α) plays an important role in liver inflammation. CD40-CD40 ligand (CD40-CD40L) is a key receptor–ligand signaling pair involved in the adaptive immune response and pathogenesis of autoimmune diseases. In mice, CD40 activation leads to sickness behavior syndrome (SBS) comprising weight loss, sleep disruption and depression, which can be blocked by administration of the TNF-inhibitor etanercept. In the present study, we assessed the extent of hepatic inflammation in mice devoid of the TNF-receptor 1 (TNFR1)-mediated signaling pathway. The TNFR1-depleted (TNFR1−/−) adult mice and their wild type littermates were given a single intra-peritoneal injection of CD40 agonist monoclonal antibody (mAb) or rat IgG2a isotope control. As described previously, TNFR1−/− mice were protected from SBS upon CD40 mAb treatment. Cd40, tnf and tnfr1 mRNA and Tnf-α peptide were increased in the liver of CD40 mAb-stimulated wild type mice. Serum alanine aminotransferase was elevated in both CD40-activated wild type and TNFR1−/− mice. TNFR1−/− mice showed much less intra-parenchymal infiltrates, hepatocellular necrosis, and perivascular clusters upon CD40 mAb activation than their wild type littermates. A gene expression microarray detected increased activity of metabolic and detoxification pathways and decreased activity of inflammatory pathways. We conclude that immune activation and development of liver inflammation in CD40L interactions depend on TNFR1-mediated signaling pathways and are counteracted by alterations in metabolic pathways.

A Novel CD16xCD33 Bispecific Killer Cell Engager (BiKE) Mediates a Double Hit For Natural Killer (NK) Cells To Target CD33+ Myelodysplastic Syndrome (MDS) Cells and Myeloid Derived Suppressor Cells (MDSCs) At All Disease Stages

MDS is a clonal heterogeneous stem cell disorder identified by pancytopenia that frequently progresses to acute myeloid leukemia (AML). The only curative treatment is hematopoietic cell transplantation (HCT), but many patients are ineligible due to advanced age and comorbidities. New therapeutic strategies are urgently needed to reduce disease burden and improve overall survival. Studies of NK cell function from MDS patients have shown diminished natural cytotoxicity against K562 targets. These NK cell defects have been ascribed to abnormalities in expression and function of the NK cell activating receptors NKG2D, NKp30, NKp46 and DNAM-1. However, the properties of the potent activating receptor CD16 (FcgRIII) and its potential for mediating targeted immunotherapy remain unexplored in MDS. Frozen pre-transplant samples from 67 MDS patients (age 19-74 years, Median=47) receiving unrelated HCT were obtained from the research sample repository of the National Marrow Donor Program and were compared to normal volunteers. NK cell degranulation, cytotoxicity and cytokine production were evaluated using published methods. We focused on CD16-induced NK cell function using a bispecific killer cell engager generated to target CD16 along with the myeloid differentiation antigen CD33 (CD16xCD33 BiKE). CD16 expression on total CD56+ MDS-NK cells was significantly lower compared to normal donors (MDS vs. normal: 63 ± 3% (n=67) vs. 89 ± 2% (n=20); p < 0.0001). However, reverse-antibody dependent cell-mediated cytotoxicity (R-ADCC) assays revealed no significant differences in CD107a (46 ± 5% (n=16) vs. 46 ± 3% (n=12)), IFN-g (6 ± 1% vs. 10 ± 2%) or TNF-a (9 ± 2% vs. 6% ± 1%) production when resting MDS-NK cells were triggered with an agonistic CD16 mAb, indicating MDS-NK cell function triggered through CD16 is intact. To evaluate the therapeutic potential of targeting CD16 in MDS patients, resting MDS-mononuclear cells were coated with 10mg/mL of the CD16xCD33 BiKE and co-cultured for 4 hours with the CD33+ target cell line HL-60. MDS-NK cell CD107a (CD16xCD33 BiKE vs. no reagent: 42 ± 2% (n=62) vs. 22 ± 1% (n=20); p < 0.0001), IFN-g (15 ± 1% vs. 3 ± 1%; p < 0.0001) and TNF-a (13 ± 1% vs. 3 ± 1%; p < 0.0001) production was significantly increased compared to the no reagent and scFv CD16 controls. Moreover, in the absence of HL-60 targets, MDS-NK cells treated with the CD16xCD33 BiKE targeted endogenous CD33+ targets, significantly enhancing degranulation (37 ± 2% vs. 17 ± 1%; p < 0.0001), IFN-g (4 ± 1% vs. 2% ± 1%; p < 0.01) and TNF-a (5 ± 1% vs. 2 ± 1%; p < 0.01) production. Recent data have shown an increase in the frequency of MDSCs, phenotypically defined as CD33+/CD11b+/CD14-/HLA-DRlo/-, during oncogenesis and in the presence of inflammatory cytokines. Indeed, in our MDS patient cohort, the frequency of MDSCs of total viable mononuclear cells was significantly higher compared to normal donors (6 ± 1% (n=64) vs. 0.1 ± 0.02% (n=18), respectively; p < 0.0001) (figure). Furthermore, CD16xCD33 BiKE-treated resting NK cells from normal donors co-cultured with cytokine-induced MDSCs generated from normal PBMCs in a 51Cr-release cytotoxicity assay successfully targeted and lysed MDSCs (CD16xCD33 BiKE vs. no reagent (E:T = 20:1): 63 ± 5% vs. 7 ± 2% [p < 0.0001, n=4]; (E:T = 6.6:1): 35 ± 3% vs. 4 ± 1% [p < 0.0001]). CD107a (48 ± 2% (n=6) vs. 4 ± 1% (n=3); p < 0.0001), IFN-g (8 ± 1% vs. 1 ± 1%; p < 0.0001) and TNF-a (8 ± 1% vs. 1 ± 1%; p < 0.0001) production was also significantly increased against MDSC targets in the presence of the CD16xCD33 BiKE, demonstrating its ability to facilitate killing of both the CD33+ MDS clone and an immune suppressor cell population. Lastly, analysis of MDS patient karyotypes in correlation with CD16xCD33 BiKE-induced MDS-NK cell CD107a, IFN-g and TNF-a production did not reveal any significant differences between favorable, intermediate or poor international prognostic scoring system (IPSS) cytogenetic scores, further demonstrating the therapeutic potential of the CD16xCD33 BiKE to induce optimal MDS-NK cell function against CD33+ targets despite disease heterogeneity. In summary, our data suggest that the CD16xCD33 BiKE may function not only against CD33+ MDS targets themselves, but also against MDSCs. This double hit approach may protect against MDS progression/transformation to AML through immune targeting and may improve blood counts by targeting MDS-MDSCs that contribute to ineffective hematopoiesis. [Display omitted] Disclosures:No relesvant conflicts of interest to declare.

Abstract CT005: T cell inflammation in the tumor microenvironment after agonist CD40 antibody: Clinical and translational results of a neoadjuvant clinical trial

Abstract Deploying CD40 activation to stimulate T cell responses upstream of immune checkpoint molecules is a novel clinical opportunity for cancer immunotherapy. Despite numerous active clinical trials with agonistic CD40 monoclonal antibodies (mAb), biological treatment effects especially treatment-related modulation of the tumor microenvironment (TME), remain poorly understood. Here, we performed a neoadjuvant clinical trial of agonistic CD40 mAb (selicrelumab) administered intravenously with or without chemotherapy (gemcitabine and nab-paclitaxel) to 16 resectable patients with pancreatic ductal adenocarcinoma (PDAC) prior to surgery followed by adjuvant chemotherapy and CD40 mAb. The toxicity profile was acceptable, including only grade 1 or 2 cytokine release syndrome and expected toxicities from chemotherapy. Disease-free survival was 13.8 months (95% CI 2.9 - 24.8 months) and median overall survival was 23.4 months (95% CI 18.0 - 28.8), with 8 patients alive at a median of 20.0 months after surgery (follow-up range 12.2 to 34.8 months). Neoadjuvant selicrelumab induced major pharmacodynamic differences in the TME, as revealed by a multiplex imaging platform auditing the immune ecosystem, compared to resection specimens from PDAC patient previously untreated or given neoadjuvant chemotherapy/chemoradiotherapy only. For tumors resected after selicrelumab, 82% (9/11) were T-cell enriched, compared to 37% (38/104) (p=0.004) of untreated tumors and 23% (93/13) of chemotherapy/chemoradiation-treated tumors (p=0.012). Moreover, for selicrelumab tumors, tumor-associated fibrosis was less, “M2” macrophages were fewer, dendritic cells were more mature, and T cells were activated and proliferative, compared to the non-selicrelumab groups. In the periphery, CD8+ and CD4+ T cells were more activated and proliferative, and serum inflammatory cytokines CXCL10 and CCL22 increased after treatment. This study provides proof-of-concept in patients that agonistic CD40 mAb alters the TME, enhances T-cell infiltration, and modulates systemic inflammatory responses. These findings inform design of next-generation CD40 clinical trials. Citation Format: Katelyn T. Byrne, Courtney B. Betts, Rosemarie Mick, Shamilene Sivagnanam, David L. Bajor, Daniel A. Laheru, E. Gabriela Chiorean, Mark H. O'Hara, Shannon M. Liudahl, Craig Newcomb, Cécile Alanio, Ana P. Ferreira, Byung S. Park, Takuya Ohtani, Austin P. Huffman, Sara A. Väyrynen, Andressa Dias Costa, Judith C. Kaiser, Andreanne M. Lacroix, Colleen Redlinger, Martin Stern, Jonathan A. Nowak, E. John Wherry, Martin A. Cheever, Brian M. Wolpin, Emma E. Furth, Elizabeth M. Jaffee, Lisa M. Coussens, Robert H. Vonderheide. T cell inflammation in the tumor microenvironment after agonist CD40 antibody: Clinical and translational results of a neoadjuvant clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT005.

Abstract 1888: Anti-CD38 interference with blood compatibility testing: Differentiating isatuximab and daratumumab via functional epitope mapping

Abstract Introduction: Two anti-CD38 monoclonal antibodies (mAb) are approved for the treatment of multiple myeloma: isatuximab (Isa) and daratumumab (Dara); binding to CD38 on red blood cells (RBCs) results in false positive agglutination on indirect antiglobulin tests (IATs). Compared to Dara, which interferes with IATs in nearly 100% of patients, Isa interfered in 67/99 (67%) patients in the Phase 3 ICARIA-MM (NCT02990338) study and 95/150 (63%) patients in the Phase 3 IKEMA (NCT0327585) study. We sought to understand differences between the binding abilities of Isa and Dara to CD38 on RBCs. Experimental Procedures: In vitro binding experiments in RBCs were performed following pretreatment of RBCs isolated from healthy donors +/- a mouse anti-human CD38 mAb (clone HB-7 or AT13/5) or a nicotinamide adenine dinucleotide (NAD) analog (irreversible CD38 inhibitor) before incubation with Isa, Dara, or control human IgG (hIgG). RBC surface bound CD38 antibodies were detected with fluorophore conjugated secondary antibodies and quantified by flow cytometry, imaging or mass spectrometry. Binding of Isa to recombinant human CD38 (rhCD38) pretreated with HB-7 or an NAD analog was quantified by Surface Plasmon Resonance (SPR). Ex vivo IAT was performed on plasma samples spiked with Isa or Dara. Results: In vitro testing in the absence of human plasma showed that incubation of RBCs with Dara but not Isa resulted in a dose-dependent increase in RBC binding. The binding specificity of Dara was demonstrated by treatment with HB-7, an anti-CD38 mAb that recognizes epitopes that overlap with Dara but not Isa binding. Flow cytometry, confocal microscopy, SPR and mass spectrometry showed HB-7 had a competitive effect on Dara binding and induced Isa to bind to CD38 without competition. Pretreatment of RBCs with another anti-CD38 mAb AT13/5 or an NAD analog also induced Isa binding to RBCs. The NAD analog is an irreversible CD38 inhibitor that stabilizes CD38 dimer conformation. SPR demonstrated that pretreatment with HB-7 or the NAD analog may enhance dimer formation increasing Isa binding affinity to rhCD38. These results indicate that CD38 conformational changes caused by anti-CD38 antibodies or the NAD analog may facilitate Isa-binding to RBC-expressing CD38. In the ex vivo IAT, except for gel testing in which both Isa and Dara were panreactive, including all other IAT methods Dara reacted with many more cells, and reactions were stronger than Isa at all concentrations tested. Conclusions: In this study, we confirmed that both Isa and Dara interfere with IATs but at different magnitudes. This reflects distinct mechanisms of CD38 binding. In the absence of human plasma, CD38 on RBCs can be directly recognized by Dara while Isa requires a cofactor, such as an anti-CD38 antibody or an NAD analog. Future studies are essential to characterize the impact of human plasma on Isa binding to RBCs-expressing CD38. Citation Format: Zhili Song, Olivier Bedel, Bailin Zhang, Joern Hopke, Gejing Deng, Sandrine Macé, Marielle Chiron, Francisco Adrian, Taro Fukao, Frank Basile, France Pirenne, Makoto Okuda, Morvarid Moayeri, Btissam Chami, Yoko Hidaka, Ashok Nambiar, Thomas Martin, Chen Zhu. Anti-CD38 interference with blood compatibility testing: Differentiating isatuximab and daratumumab via functional epitope mapping [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1888.

Efficacy and Safety of First-Line Therapy with Chlorambucil, Rituximab and Individualized Dose Lenalidomide in FCR-Unfit Patients with Advanced Chronic Lymphocytic Leukemia (CLL): A Phase I/II HOVON Trial

Efficacy and Safety of First-Line Therapy with Chlorambucil, Rituximab and Individualized Dose Lenalidomide in FCR-Unfit Patients with Advanced Chronic Lymphocytic Leukemia (CLL): A Phase I/II HOVON Trial