CD20 Antibody Research Articles

A nondepleting anti-CD19 antibody impairs B cell function and inhibits autoimmune diseases.

B cells contribute to multiple aspects of autoimmune disorders, and B cell-targeting therapies, including B cell depletion, have been proven to be efficacious in treatment of multiple autoimmune diseases. However, the development of novel therapies targeting B cells with higher efficacy and a nondepleting mechanism of action is highly desirable. Here we describe a nondepleting, high-affinity anti-human CD19 antibody LY3541860 that exhibits potent B cell inhibitory activities. LY3541860 inhibits B cell activation, proliferation, and differentiation of primary human B cells with high potency. LY3541860 also inhibits human B cell activities in vivo in humanized mice. Similarly, our potent anti-mCD19 antibody also demonstrates improved efficacy over CD20 B cell depletion therapy in multiple B cell-dependent autoimmune disease models. Our data indicate that anti-CD19 antibody is a highly potent B cell inhibitor that may have potential to demonstrate improved efficacy over currently available B cell-targeting therapies in treatment of autoimmune conditions without causing B cell depletion.

Preclinical anti-tumour activity of HexaBody-CD38, anext-generation CD38 antibody with superior complement-dependent cytotoxic activity.

HexaBody®-CD38 (GEN3014) is a hexamerization-enhanced human IgG1 that binds CD38 with high affinity. The E430G mutation in its Fc domain facilitates the natural process of antibody hexamer formation upon binding to the cell surface, resulting in increased binding of C1q and potentiated complement-dependent cytotoxicity (CDC). Co-crystallization studies were performed to identify the binding interface of HexaBody-CD38 and CD38. HexaBody-CD38-induced CDC, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), trogocytosis, and apoptosis were assessed using flow cytometry assays using tumour cell lines, and MM patient samples (CDC). CD38 enzymatic activity was measured using fluorescence spectroscopy. Anti-tumour activity of HexaBody-CD38 was assessed in patient-derived xenograft mouse models invivo. HexaBody-CD38 binds a unique epitope on CD38 and induced potent CDC in multiple myeloma (MM), acute myeloid leukaemia (AML), and B-cell non-Hodgkin lymphoma (B-NHL) cells. Anti-tumour activity was confirmed in patient-derived xenograft models invivo. Sensitivity to HexaBody-CD38 correlated with CD38 expression level and was inversely correlated with expression of complement regulatory proteins. Compared to daratumumab, HexaBody-CD38 showed enhanced CDC in cell lines with lower levels of CD38 expression, without increasing lysis of healthy leukocytes. More effective CDC was also confirmed in primary MM cells. Furthermore, HexaBody-CD38 efficiently induced ADCC, ADCP, trogocytosis, and apoptosis after Fc-crosslinking. Moreover, HexaBody-CD38 strongly inhibited CD38 cyclase activity, which is hypothesized to relieve immune suppression in the tumour microenvironment. Based on these preclinical studies, a clinical trial was initiated to assess the clinical safety of HexaBody-CD38 in patients with MM. Genmab.

KAPPA MYELOMA ANTIGEN (KMA) AND LAMBDA MYELOMA ANTIGEN (LMA) ARE EXPRESSED ON B CELL MALIGNANCIES AND ARE PROMISING NOVEL TARGETS ON MALIGNANT PLASMA CELLS

Introduction: The kappa myeloma antigen (KMA) and the lambda myeloma antigen (LMA) are found on the surface of malignant plasma cells (PCs) in multiple myeloma (MM), other plasma cell dyscrasia patient bone marrow (BM) samples, human myeloma cell lines and in patient BM samples from plasmacytomas, lymphoplasmacytoid and B cell lymphoproliferative disorders (BLPD). KMA and LMA are not present on normal B cells in BM but are found on occasional mononuclear cells in tonsillar tissue and secondary mucosal lymphoid tissue. KappaMab is a therapeutic antibody that specifically targets KMA and a phase IIb study in combination with lenalidomide and dexamethasone has recently been completed. In early clinical trials, KappaMab was shown to have an excellent safety profile, with low-grade infusion-related reactions and no haematological toxicities. Two human therapeutic LambdaMabs (10B3 and 7F11) bind specifically to LMA expressed only on malignant cells in lambda restricted B cell diseases. Here we compare the expression of KMA and LMA to that of BCMA in MGUS, newly diagnosed MM and relapsed, refractory (RRMM) myeloma BM samples. We describe the difference in phenotype between KMA and LMA expressing malignant PCs and their enrichment in RRMM compared to early disease stages. Methods: Patient BM samples (κ = 70 and λ = 40) were analysed using multiparametric FCM immunophenotyping with APC labelled LambdaMab (10B3) and KappaMab Fab’2 fragments, CD38, CD138, CD269 (BCMA), CD319 (SLAM F7), CD56 and CD45 monoclonal antibodies. PCs were identified by initial gating using CD38 and CD138. LMA and KMA expression was determined and compared with the other cell markers. Results: KMA was expressed on the majority of kappa restricted BM PCs from MGUS through to RRMM, on lymphoplasmacytoid cells in 2 out of 3 BM samples and in all 4 plasmacytoma BM samples. LMA was expressed on the surface of BM PCs in lambda restricted MGUS through to RRMM and in all 4 AL amyloidosis BM samples. KMA and LMA antigen densities were both higher than BCMA in the RRMM population: KMA mean 1899 (range 468–7943) versus BCMA mean 1426 (range 350-2630) and LMA mean 2439 (range 263–6664) versus BCMA mean 817 (range 537–1065) molecules of PE/cell. Conclusions: Although there are smaller numbers of samples from other B cell malignancies such as lymphoplasmacytoid, amyloidosis and BLPDs, we have demonstrated that KMA or LMA are expressed on malignant BM PCs. In the myeloma cases, the antigen density of both KMA and LMA was increased compared to BCMA on RRMM BM PCs, which suggests a clonal advantage of KMA and LMA expressing plasma cells that are resistant to standard of care treatments. These data suggest that KappaMab and LambdaMabs have therapeutic potential in RRMM where the combination of high antigen density and restriction of the target antigen to the malignant subpopulation of PCs may confer clinical benefit by retaining normal plasma cells following treatment. Keywords: Immunotherapy, Multiple Myeloma Conflicts of interests pertinent to the abstract D. Gottlieb Consultant or advisory role David sits on the Scientific Advisory Board for HaemaLogiX Ltd. R. Dunn Employment or leadership position: Employee at HaemaLogiX Ltd. Stock ownership: Yes

Sequential salvage chemotherapy and lintuzumab-Ac225 results in deep responses and prolonged survival in adverse risk relapsed/refractory AML and in AML patients that received prior venetoclax therapy.

e19030 Background: Lintuzumab-Ac225 is a humanized CD33 antibody conjugated to an alpha emitting isotope, Ac225, delivering high energy radiation to CD33 expressing AML blasts. Preclinical studies indicate Lintuzumab-Ac225 also depletes the anti-apoptotic protein MCL-1, which is a known resistance factor in BCL2-inhibitor (venetoclax (Ven)) exposed patients (pts). We performed a Phase I study where Lintuzumab-Ac225 was administered after salvage chemotherapy, and this study demonstrated safety and encouraging response rates in R/R AML. Herein, we report results of high-risk patients who enrolled to this study. Methods: R/R AML pts deemed fit, and with CD33 expression on >25% of blasts were enrolled onto a Phase I study, Lintuzumab-Ac225 in Combination with Cladribine + Cytarabine + Filgrastim + Mitoxantrone (CLAG-M). Induction consisted of G-CSF, 300mcg/d on D1-6, cladribine 5mg/m2 on D2-6, cytarabine 2g/m2 on D2-6, and mitoxantrone 10mg/m2 on D2-4. Lintuzumab-Ac225 was administered once on D8 ±1 day. The trial enrolled to 4 cohorts, with Lintuzumab-Ac225 at doses ranging from 0.25 uCi/kg to 1.0 uCi/kg. We defined high-risk pts as those who met ELN 2019 criteria for adverse risk disease, or pts who received a Ven combination prior to trial enrollment. Response is descriptively reported, and KM estimator was used to report overall survival (OS). Results: 19 pts on trial met high-risk criteria. Of these, 16 pts had ELN adverse risk disease, a TP53 mutation was detected in 75% (n=12) of pts. Median age was 65 yrs, pts received a median 2 lines of prior therapy, including prior allo-HCT in 58% (n=11), and prior Ven combination in 68% (n=13) of pts. In high-risk pts, 42% (n=8) of pts achieved CRc (CR+CRi), 16% (n=3) achieved MLFS, for an overall response rate (ORR) of 58%. Among evaluable pts achieving CRc, 71% achieved flow MRD negativity. Median OS was 7.4 months. Table reports results in select subgroups. Prior to treatment initiation, 7 pts were deemed eligible for first HCT. 4 (57%) were successfully able to bridge to HCT. Median survival among transplanted pts was 28 months. Post-HCT, one pt died due to GVHD, one due to relapse. Conclusions: CLAG-M in combination with Lintuzumab-Ac225 yielded significantly better clinical outcomes in high-risk populations, particularly in pts previously treated with Ven combinations (median OS 13.8 mo.) and in pts with TP53 mutations (median OS 7.3 mo.), for which recent studies have reported a survival of 4 months or less. The results support further advance in late-stage clinical development of Lintuzumab-Ac225 in combination with CLAG-M in R/R AML, especially in high-risk pts. Clinical trial information: NCT03441048 . [Table: see text]

A phase 1b/2 study of pivekimab sunirine (PVEK, IMGN632) in combination with venetoclax/azacitidine or magrolimab for patients with CD123-positive acute myeloid leukemia (AML).

TPS7073 Background: Despite improved outcomes with azacitidine (AZA) and venetoclax (VEN) in frontline unfit AML (CR 37% and CR/CRi 66%), long-term survival for these patients remains inadequate (mOS < 15m, DiNardo NEJM 2020). CD123 is expressed on the majority of AML blasts and leukemic stem cells while minimally expressed on normal hematopoietic stem cells (Kovtun Blood Adv 2018). Pivekimab sunirine (PVEK, IMGN632) is an antibody-drug conjugate (ADC) comprising a high-affinity CD123 antibody, cleavable linker, and an indolinobenzodiazepine pseudodimer (IGN) payload. The IGN payload alkylates DNA and causes single strand breaks without crosslinking. IGNs are designed to have high potency against tumor cells, while demonstrating less toxicity to normal marrow progenitors than other DNA-targeting payloads. Clinical data in relapsed/refractory (R/R) AML (Daver ASH 2022) supported the continued clinical exploration of the PVEK+AZA+VEN triplet. The current enrolling dose expansion cohorts (Regimen C; Cohorts 1 and 2) in frontline AML will evaluate antileukemia activity and MRD levels in the combination of PVEK+AZA+VEN. Regimen E will enroll patients with R/R AML to evaluate the safety and antileukemia activity of PVEK in combination with magrolimab. Methods: This is an open-label, multicenter, Phase 1b/2 study of PVEK administered in combination regimens in patients with frontline and R/R CD123-positive AML. Patients will receive the established recommended phase 2 dose (RP2D) of PVEK 0.045 mg/kg IV, as a < 30-minute outpatient infusion. In Regimen C, patients will receive PVEK 0.045 mg/kg IV on D7 + azacitidine 75 mg/m2 SC or IV daily on days 1 to 7, and venetoclax up to 400 mg PO daily for at least 14 days (Cohort 1) or for up to 28 days (Cohort 2) in a 28-day cycle. In Regimen E, R/R AML patients will receive PVEK 0.045 mg/kg IV on D1 of the 28-day cycle in combination with magrolimab at 30 mg/kg Q2W, after the standard ramp up schedule. Regimen C will enroll adults with untreated AML, CD123+ by flow cytometry or IHC and no prior HMA treatments. The primary endpoints for Regimen C are composite CR rate, overall response rate (ORR), duration of remission (DOR), MRD levels and key secondary endpoints are safety, PK and immunogenicity. Regimen E will enroll adult patients with relapsed or refractory CD123+ AML with up to 2 prior lines of therapy. The primary endpoint for Regimen E is dose limiting toxicities (DLTs) and key secondary endpoints are safety, antileukemic activity, PK and immunogenicity. The PVEK+AZA+VEN triplet (Regimen C) is currently enrolling frontline unfit patients across sites in France, Germany, Italy, Spain, UK and USA. The PVEK+Magro doublet (Regimen E) is planned to be open for enrollment mid-2023 at sites in the USA. Clinical trial information: NCT04086264 .

Abstract ND01: ABBV-319: A first-in-class Glucocorticoid Receptor Modulator (GRM) agonist ADC for the treatment of B-cell malignancies

Abstract ABBV-319 is a novel CD19-targeting multi-modal antibody drug conjugate (ADC) for the treatment of B-cell malignancies. The ADC is engineered to have 3 distinct mechanisms of action to maximize anti-cancer activity while reducing systemic toxicities: (1) targeted GRM payload delivery, (2) signaling inhibition of CD19/BCR and (3) enhanced ADCC via afucosylation. Unlike traditional cytotoxic ADCs, the GRM payload is a high potency agonist (sub nM EC50) of the intracellular glucocorticoid receptor (GR) which then traffics to the nucleus resulting in sustained GR-driven transcriptional events leading to cancer cell death. CD19 is a clinically validated B-cell target (approved antibodies, ADCs, CARTs) known to be highly expressed even following multiple lines of therapy, making it an appropriate target for this novel approach. The CD19 antibody in ABBV-319 is an optimized high affinity IgG1 that can inhibit CD19/BCR downstream signaling events including AKT, leading to inhibition of growth in certain B-cell lines. Given ADCC/ADCP is a highly effective mechanism in B-cell malignancies (e.g., rituximab, tafasitamab) the CD19 antibody was engineered to have increased FcγR binding via afucosylation, that retains its enhanced ADCC activity as a GRM drug conjugate. By engineering the molecule in this way, the multi-modal ADC is not solely reliant on one mechanism of action for activity and therefore has the potential for broader activity. Sustained regressions/cures have been observed in multiple B-cell malignancy xenograft model systems (cell-line, PDX, humanized) after a single ADC dose that compares favorably against approved benchmark antibodies, systemic steroids and ADCs. ABBV-319 targets clinically validated mechanisms of action that are known to combine well with multiple standard-of-care agents. This combination potential together with its favorable safety profile, suggests ABBV-319 will be a highly effective combination treatment for B-cell malignancies. ABBV-319 was well tolerated in cynomolgus monkey studies with favorable drug-like properties and antibody-like pharmacokinetics, enabling a high mg/kg clinical starting dose. A first in-human Phase 1 clinical trial evaluating ABBV-319 in B-cell malignancies (DLBCL, FL, CLL) was recently initiated in late 2022 (clinicaltrials.gov, NCT05512390). Citation Format: James W. Purcell. ABBV-319: A first-in-class Glucocorticoid Receptor Modulator (GRM) agonist ADC for the treatment of B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr ND01.

Abstract 5786: Spatial relationships in the tumor microenvironment predict response to immune checkpoint inhibitors in urothelial and head and neck cancer

Abstract Background: Immune checkpoint inhibitors (ICI) can achieve remarkable clinical responses in urothelial cancer (UC). However, it remains unclear which aspects of the tumor microenvironment (TME) determine a patient’s response. The TME is usually characterized by immune cell density, which ignores cells’ spatial relationships relative to each other. Methods: Using multiplex immunofluorescence data (PanCK, CD20, CD68, CD3, CD8, and FoxP3 antibody panel) of 24 pre-ICI UC transurethral resections retrieved from the NABUCCO trial (NCT03387761), we spatially profiled cancer cells, macrophages, B-cells, and distinct T-cell populations. We first quantified the TME immune cell densities at the tumor and stroma tissue compartments. We then quantified the TME spatial relationships with a novel approach based on fitting a Weibull function to the first nearest neighbor (1-NN) distance distribution, allowing us to uniquely summarize spatial relationships with two parameters (Weibull approach). We compared this approach to conventional techniques (G-functions) that rely upon a predefined distance threshold. We performed a simulation study to identify sources of variation in the spatial relationship parameters. Lastly, we associated the TME parameters with ICI (ipilimumab + nivolumab) response. Results: Unlike the Weibull approach, the G-function quantifications manifested a variable effect size and statistical power in association studies because of its distance threshold dependence. We found that variation in density affected the spatial relationship metrics of rare cell types (i.e., B-cells) but not of abundant cell types (i.e., cancer cells). The spatial relationship metrics from the Weibull approach outperformed immune cell density in ICI response prediction. Specifically, immune cell density did not discriminate between ICI response groups (FDR>10%). In contrast, the spatial relationship between either CD8+ T-cells or macrophages to their closest cancer cell did associate with response to ICI (FDR=1%). Furthermore, non-responding tumors were characterized by CD8+ T-cells close to B-cells (FDR=9%). Importantly, we validated the association between proximity and response from CD8+ T-cells to cancer cells (FDR=1%) and from macrophages to cancer cells (FDR=1%) using data from 25 pre-ICI head and neck squamous cell carcinoma tumors from the IMCISION trial (NCT03003637, arm B, ipilimumab + nivolumab). Conclusion: We created a framework to quantify, study, interpret and analyze spatial relationships in the TME and illustrated its superior clinical relevance compared to density metrics for predicting ICI treatment response. Our findings emphasize the importance of spatial relationships in the TME for response and suggest that proximity between either macrophages or CD8+ T-cells to cancer cells are candidate biomarkers for ICI response. Citation Format: Alberto Gil-Jimenez, Nick van Dijk, Yoni Lubeck, Maurits L. van Montfoort, Dennis Peters, Erik Hooijberg, Annegien Broeks, Joris L. Vos, Charlotte L. Zuur, Bas van Rhijn, Daniel J. Vis, Michiel S. van der Heijden, Lodewyk F. Wessels. Spatial relationships in the tumor microenvironment predict response to immune checkpoint inhibitors in urothelial and head and neck cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5786.

Abstract 4421: Targeting myeloid-derived suppressor cells with actinium-225 lintuzumab, a CD33 antibody radioconjugate to enhance antitumor immunity

Abstract Introduction: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid lineage cells with potent immunosuppressive activity found enriched in cancer patients. Crosstalk between MDSCs and the tumor microenvironment can promote tumor immune evasion and thus there is growing interest in developing therapeutic approaches to intervene in the MDSC suppressive function. Both monocytic-MDSC (M-MDSC) and granulocytic-MDSC (G-MDSC) subpopulations express the cell surface CD33 myeloid marker. For well-defined cell surface markers like CD33, there is considerable interest in the use of radionuclides as therapeutic payloads, particularly α-particle emitters such as actinium-225 (225Ac) since they deliver substantially higher decay energies over a much shorter distance than β-emitters, rendering them more suitable for precise, potent, and efficient target cell killing while minimizing toxicity to surrounding bystander cells. Actimab A, the anti-CD33 antibody lintuzumab armed with the 225Ac radioisotope (CD33 ARC), is currently being evaluated in R/R AML and has demonstrated significant anti-leukemic activity in Phase 1/2 clinical trials. We therefore hypothesized that MDSCs can be directly targeted by the CD33 ARC. Hence, we evaluated the therapeutic potential of the CD33 ARC to deplete MDSCs through preclinical studies in vitro and in vivo with humanized mouse model. Methods: CD33 ARC was generated by conjugating lintuzumab with p-SCN-Bn-DOTA and subsequently radiolabeled with 225Ac. Primary MDSCs were isolated from cancer patient peripheral blood or healthy donor PBMCs. The specific binding of CD33 ARC to CD33 positive MDSCs and the decreased viability of MDSCs in response to treatment in vitro was characterized by immunophenotyping using flow cytometry. The therapeutic efficacy of CD33 ARC to deplete MDSC in vivo was evaluated in human CD34 reconstituted humanized NOG-EXL mice. Results: CD33 positive MDSCs (M-MDSCs and G-MDSCs) were identified in human cancer peripheral blood samples, including colorectal and lung. Significantly more MDSCs were found in the peripheral blood of cancer patients in comparison to healthy donors. CD33 ARC treatment of human cancer MDSCs induced a potent dose-dependent reduction in MDSC viability leading to increased depletion of MDSCs. Furthermore, in the humanized NOG-EXL mouse model, CD33 ARC therapy demonstrated in vivo activity of CD33 ARC to deplete human MDSCs. Conclusions: In this study, we demonstrate CD33 ARC alpha targeted radiotherapy depletes human CD33 positive immune suppressing MDSCs present in multiple cancer types, to enhance antitumor immunity. These findings present a translatable strategy that supports further evaluation of 225Ac lintuzumab as a MDSC targeting agent to improve the efficacy of antitumor therapies. Citation Format: Amanda Chin, Mary Chen, Jason Li, Megan McCloskey, Caroline Jennings, Le-Cun Xu, Monideepa Roy, Denis Beckford, Helen Kotanides. Targeting myeloid-derived suppressor cells with actinium-225 lintuzumab, a CD33 antibody radioconjugate to enhance antitumor immunity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4421.

Abstract 6329: Preclinical study of CD19 detection methods using monoclonal antibodies post tafasitamab treatment

Abstract Introduction Tafasitamab (TAFA), an Fc-enhanced antibody immunotherapy against CD19 received accelerated/conditional approval in relapsed or refractory diffuse large B cell lymphoma (R/R DLBCL) in combination with lenalidomide. Several other CD19 targeting therapies including CAR Ts are now available for R/R DLBCL treatment warranting evaluation of potential therapeutic sequencing strategies. In this context, CD19 expression status may be assessed on patient samples post TAFA treatment. However, methods for detecting CD19 using monoclonal antibodies (mAbs) following TAFA treatment are not standardized which may lead to data misinterpretations including confusion of CD19 epitope masking with CD19 loss or downregulation. Aim This study evaluated various CD19 detection methods following TAFA treatment using comprehensive panels of available flow cytometry (FC) and immunohistochemistry (IHC) anti-CD19 mAbs, routinely used in laboratory practice. Methods FC and IHC competition assays were used on TAFA pre-treated CD19+ cell lines to differentiate non-competing from competing mAbs, the latter resulting in epitope masking by TAFA. Bound TAFA was removed from CD19+ cells using an acidic dissociation protocol. Antibody affinities for CD19 were determined using Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI). Results All but one of the 8 mAbs tested in FC competed with TAFA and their affinities for CD19 were shown to be lower than that of TAFA. The non-competing clone OTI3B10, reported to recognize a linear epitope of CD19 (1), bound to only one of three tested CD19+ cell lines, which rendered it unsuitable for reliable FC detection of CD19. In contrast, several clones could detect CD19 on TAFA pre-treated samples using IHC. To circumvent TAFA masking in FC, we successfully unmasked CD19 via acidic dissociation and similar CD19 levels on TAFA pre-treated as on untreated cells could be subsequently detected by a competing CD19 antibody (HIB19). Conclusion Our study provides an overview of commonly used mAb tools for CD19 detection post TAFA treatment, some of which may be used in routine clinical practice. Our findings reveal that CD19 expression post TAFA could be detected by IHC but lack of TAFA non-competing anti-CD19 antibodies for FC poses a risk of mistaking CD19 masking by TAFA with CD19 loss or downregulation. Prior acidic dissociation is critical in the staining protocol to avoid data misinterpretations. 1. Klesmith et al, 2019, Biochem. Citation Format: Kristina M. Ilieva, Markus Eberl, Jan Jaehrling, Carmen Ginzel, Britta Voland, Michaela Zankl, Andrea Polzer, Derek Blair, Rainer Boxhammer, Liang-Chuan Wang, Diana Alvarez Arias, Christina Heitmüller. Preclinical study of CD19 detection methods using monoclonal antibodies post tafasitamab treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6329.

Abstract 2655: Anti-CD19 antibody-drug conjugate therapy in B cell non-Hodgkin lymphoma

Abstract Previously, we have shown that overexpression of Polo-like kinase (PLK)-1 is associated with poor clinical outcomes in patients with aggressive B cell lymphomas. PLK-1 selective inhibitor, Volasertib (BI 6727), has a potent anti-tumor effect in aggressive B cell lymphomas. Unfortunately, Volasertib was withdrawn in Phase III clinical trials due to severe myelosuppression. In this study, we developed a Volasertib antibody-drug conjugate (V-ADC) using -CD19 antibody Inebilizumab to increase the targeting specificity for B-cell lymphoma cells and minimize the side effects of Volasertib. However, we found that V-ADC treatment exhibited little cytotoxic effects, comparing to free Volasertib treatment in Z138 cells, a mantle cell lymphoma (MCL) cell line. We hypothesized that the expression level/density of CD19 on B-lymphoma cells as well as its gene mutations may affect V-ADC binding affinity required for its endocytosis. We, therefore, established Z138 cell lines with overexpression of either wild-type (WT) or mutant (Mut) CD19. The cell viability at 48h post 10nM V-ADC treatment was determined to be 90%, 61%, and 69% in parent, WT, and Mut CD19-overexpressing Z138 cells, respectively. In comparison, the cell viability with 10nM free volasertib treatment ranged from 3-30% in these cell lines. Therefore, other factors, in addition to CD19 expression, may have also played an important role in V-ADC induced cytotoxicity. Upon endocytosis, the V-ADC will enter the endolysosome where cathepsin, a lysosomal enzyme, cleaves the linker between Volasertib and Inebilizumab and releases free Volasertib into the cells. Our previous works have shown that a starvation condition (culture media with 1% FBS) increased lysosomal cathepsin activity, comparing to the culture media with 10% FBS. Herein, we found that upon treatment with 10nM V-ADC, CD19-overexpressing Z138 cells cultured in media with 1% FBS exhibited markedly increased apoptotic cell death comparing to cells in media with 10% FBS (64% vs 41%, respectively). Furthermore, WT and Mut CD19-overexpressing Z138 cells exhibited 2.8- and 2.4-folds of apoptotic cell death compared to control Z138 cells at 48h post 10nM V-ADC treatment with 1% FBS. The cell apoptosis occurred via upregulation of caspase 3, but not caspase 9, with induction of cleavage of poly (ADP-ribose) polymerase (PARP) and reduction of mitochondria membrane potential. In conclusion, starvation and lysosomal cathepsin activation increased V-ADC-induced apoptotic cell death in CD19 overexpression Z138 cell lines. We are actively investigating the in vivo therapeutic effects of V-ADC in patient-derived xenograft (PDX) animal models of MCL and other aggressive B-cell lymphomas. Citation Format: Chieko Saito, Chengfeng Bi, Kai Fu. Anti-CD19 antibody-drug conjugate therapy in B cell non-Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2655.

Dual targeting of CD19 and CD22 against B-ALL using a novel high-sensitivity aCD22 CAR

CAR Tcells recognizing CD19 effectively treat relapsed and refractory B-ALL and DLBCL. However, CD19 loss is a frequent cause of relapse. Simultaneously targeting a second antigen, CD22, may decrease antigen escape, but is challenging: its density is approximately 10-fold less than CD19, and its large structure may hamper immune synapse formation. The characteristics of the optimal CD22 CAR are underexplored. We generated 12 distinct CD22 antibodies and tested CARs derived from them to identify a CAR based on the novel 9A8 antibody, which was sensitive to low CD22 density and lacked tonic signaling. We found no correlation between affinity or membrane proximity of recognition epitope within Ig domains 3-6 of CD22 with CART function. The optimal strategy for CD19/CD22 CART co-targeting is undetermined. Co-administration of CD19 and CD22 CARs is costly; single CARs targeting CD19 and CD22 are challenging to construct. The co-expression of two CARs has previously been achieved using bicistronic vectors. Here, we generated a dual CART product by co-transduction with 9A8-41BBζ and CAT-41BBζ (obe-cel), the previously described CD19 CAR. CAT/9A8 CART eliminated single- and double-positive target cells invitro and eliminated CD19- tumors invivo. CAT/9A8 CART is being tested in a phase I clinical study (NCT02443831).

B cell targeted therapies in inflammatory autoimmune disease of the central nervous system.

Cumulative evidence along several lines indicates that B cells play an important role in the pathological course of multiple sclerosis (MS), neuromyelitisoptica spectrum disorders (NMOSD) and related CNS diseases. This has prompted extensive research in exploring the utility of targeting B cells to contain disease activity in these disorders. In this review, we first recapitulate the development of B cells from their origin in the bone marrow to their migration to the periphery, including the expression of therapy-relevant surface immunoglobulin isotypes. Not only the ability of B cells to produce cytokines and immunoglobulins seems to be essential in driving neuroinflammation, but also their regulatory functions strongly impact pathobiology. We then critically assess studies of B cell depleting therapies, including CD20 and CD19 targeting monoclonal antibodies, as well as the new class of B cell modulating substances, Bruton´s tyrosinekinase (BTK) inhibitors, in MS, NMOSD and MOGAD.

Nano-LYTACs for Degradation of Membrane Proteins and Inhibition of CD24/Siglec-10 Signaling Pathway.

Lysosome-targeting chimeras (LYTACs) are an emerging therapeutic modality that effectively degrade cancer cell membranes and extracellular target proteins. In this study, a nanosphere-based LYTAC degradation system is developed. The amphiphilic peptide-modified N-acetylgalactosamine (GalNAc) can self-assemble into nanospheres with a strong affinity for asialoglycoprotein receptor targets. They can degrade different membranes and extracellular proteins by linking with the relevant antibodies. CD24, a heavily glycosylated glycosylphosphatidylinositol-anchored surface protein, interacts with Siglec-10 to modulate the tumor immune response. The novel Nanosphere-AntiCD24, synthesized by linking nanospheres with CD24 antibody, accurately regulates the degradation of CD24 protein and partially restores the phagocytic function of macrophages toward tumor cells by blocking the CD24/Siglec-10 signaling pathway. When Nanosphere-AntiCD24 is combined with glucose oxidase, an enzyme promoting the oxidative decomposition of glucose, the combination not only effectively restores the function of macrophages in vitro but also suppresses tumor growth in xenograft mouse models without detectable toxicity to normal tissues. The results indicate that GalNAc-modified nanospheres, as a part of LYTACs, can be successfully internalized and are an effective drug-loading platform and a modular degradation strategy for the lysosomal degradation of cell membrane and extracellular proteins, which can be broadly applied in the fields of biochemistry and tumor therapeutics.

Neoplastic Disorders of Visual Apparatus: Advances and Management

Purpose: Ocular neoplasms, both primary and metastatic, may present with visual disturbance or vision loss and often are asymptomatic. Clinical examination may demonstrate leukocoria, abnormal pupillary light reflex, or a mass lesion with or without retinal detachment or hemorrhage. Retinoblastoma in children and uveal melanoma and ocular metastases in adults are the most important ocular malignant neoplasms referred for imaging to aid with diagnosis and staging. Familiarity with their common imaging appearances, the common patterns of spread, and the diagnostic findings of greatest concern to the optometrist or ocular oncologist will enhance accuracy of imaging interpretation.
 Methodology: Clinical examination and imaging using B-scan ultrasound, A-scan ultrasound, fluorescein angiography, computed tomography and magnetic resonance imaging have complementary roles in ocular tumor staging and treatment assessment.
 Findings: Ocular neoplasm tumors are relatively rare but require unique diagnostic and treatment considerations given the functional importance of the eye and periocular structures and their unique metastatic behavior. In the following paper, a major malignant tumor of the ocular adnexa including the eyelid, conjunctiva and orbit will be reviewed. Frozen section control of the margins and, in selected cases, Mohs microsurgery have decreased the recurrence rate in malignant eyelid tumors. Intraoperative cryotherapy and postoperative topical mitomycin C have similarly contributed to better surgical outcomes in conjunctival malignant tumors including squamous cell carcinoma and malignant melanoma. Immunotherapy with CD20 antibodies is a developing treatment in Ocular neoplasm lymphomas.
 Unique Contribution to Theory, Practice and Policy: Optometrists by the new advances in clinical optometry training/research, and specialists in occuloplasty/ocularistry form part of the team for a multidisciplinary approach in the clinical management of neoplastic disorders of visual apparatus.

Transferring measurable residual disease measurement in pediatric acute lymphoblastic leukemia from quantitative real-time PCR to digital droplet PCR.

Since the measurement of measurable residual disease (MRD) is part of clinical routine examination for children affected with acute lymphoblastic leukemia (ALL), continuous efforts are made to improve its method, applicability and accuracy. Whereas quantitative real-time polymerase chain reaction (qPCR) is considered as the gold standard for MRD detection and endowed with international guidelines for implementation and evaluation, these do not yet exist for digital droplet PCR (ddPCR). However, advantages are seen in droplet partitioning for MRD measurement to allow absolute quantification without depending on reference samples. In this study, 17 MRD targets of nine patients with childhood B-ALL were analyzed with qPCR and ddPCR, respectively. All patients were assigned to high risk group and had hematopoietic stem cell transplantation and CD19 antibody therapy for relapse prevention. Starting with the sequences and guidelines of qPCR and optimizing the protocol for ddPCR, the MRD targets could also be measured precisely with this novel method, using the same primer and probe sets as for qPCR. The already established MRD protocol of qPCR could be transferred to ddPCR and all 17 MRD targets were measured in dilution series reaching comparable Limit of detection levels with both PCR methods. With a given qPCR protocol and some experience in conventional MRD monitoring, it is conceivable to transfer the procedure of MRD measurement to ddPCR technology. Our data is in line with other studies which are summarized and discussed here as well to facilitate the transfer of MRD diagnostics to ddPCR.

QuPath Analysis for CD30+ Cutaneous T-Cell Lymphoma.

Mycosis fungoides is the most common subtype of cutaneous T-cell lymphoma, in which the expression of cluster of differentiation 30 (CD30)+ subtype can now be treated with the CD30 antibody conjugate brentuximab vedotin. Diagnostic methods are based on immunohistochemical (IHC) staining followed by manual assessment by pathologists, which is always a subjective calculation. QuPath, an open-source software for digital pathology image analysis, satisfies the requirements of objective approaches. Ten samples from mycosis fungoides patients with CD30 expression at different stages were stained for CD3 and CD30 by IHC staining, scanned, and quantitative analysis was performed using QuPath (version 2.1). Each slide was independently assessed by 3 board-certified dermatopathologists. Individual estimates for CD30+/CD3+ cells varied among the individual histopathologists (mean coefficient of variation, 0.46; range, 0-0.78). QuPath analysis showed excellent separation between the positively stained cells for CD3 and CD30 IHC and other cells and tissue structures, although the results correlated strongly with the respective mean estimates of the 3 histopathologists (Pearson-R 0.93). The results show a high interobserver variability evaluation of IHC markers, although quantitative image analysis offer a significant advantage for comparison. This is not only relevant for clinical routine but also especially critical in therapeutic studies addressing targeted molecules.

Daratumumab as a novel treatment option in refractory ITP

Primary immune thrombocytopenia (ITP) in adult patients typically presents as a repeatedly relapsing disease in need of multiple lines of therapy. Here we report the clinical courses of two patients, an 82-year-old female and a 54-year-old male, with primary ITP after multiple relapses and exhausted standard therapies, which we treated with the myeloma-licensed anti-CD38 monoclonal antibody daratumumab in an off-label setting. Daratumumab is known to target preferentially plasmablasts, short-lived plasma cells and long-lived plasma cells, with the latter being the major source of antiplatelet autoantibodies. Noteworthy, rituximab, a CD20 antibody, targets earlier steps in B-cell ontogenesis, thereby indirectly decreasing plasmablasts and short-lived plasma cells, but to a lesser extent long-lived plasma cells, which tend to persist after rituximab treatment. Several single-patient reports and case series have demonstrated successful treatment with daratumumab in ITP, autoimmune thrombocytopenia in Evans syndrome as well as other cytopenias or pure red cell aplasia after allogeneic stem cell transplantation or in congenital diseases, systemic lupus erythematodes and cold agglutinin disease. Our first patient with isolated primary ITP rapidly and lastingly responded to daratumumab plus tapered steroids, with platelet counts above 50 × 109/L within weeks and subsequently even stably within the normal range. Despite no objective response observed in the second patient, a lasting clinical stabilization was achieved. As the underlying mode of action, we hypothesize here daratumumab to effectively target long-lived plasma cells as the source of ITP-mediating autoantibodies, and suggest broader clinical evaluation of daratumumab in this potential indication.

Infection risks and prevention in patients with multiple myeloma

Multiple myeloma (MM) is a hematological malignancy characterized by aberrant clonal plasma cells in the bone marrow. Despite significant advances in the treatment of MM, infection remains a main cause of death. MM patients have an increased risk of infection compared to their healthy counterparts due to several factors related to underlying disease, advanced age, comorbidities, and MM treatment. MM patients are at high risk of infection during the first 3 months after diagnosis and during relapse. Anti-myeloma drugs frequently result in severe immunosuppression and increased risk of infection. Proteasome inhibitors deplete T cells, lenalidomide and pomalidomide induce neutropenia, and CD38 antibodies reduce B cell function. To prevent infection in MM patients, we should take appropriate action by medical prophylaxis and vaccination.

T cell autoimmunity and immune regulation to desmoglein 3, a pemphigus autoantigen.

Pemphigus is a life-threatening autoimmune bullous disease mediated by anti-desmoglein IgG autoantibodies. Pemphigus is mainly classified into three subtypes: pemphigus vulgaris, pemphigus foliaceus, and paraneoplastic pemphigus. The pathogenicity of autoantibodies has been extensively studied. Anti-human CD20 antibody therapy targeting B cells emerged as a more effective treatment option compared to conventional therapy for patients with an intractable disease. On the other hand, autoreactive T cells are considered to be involved in the pathogenesis based on the test results of human leukocyte antigen association, autoreactive T cell detection, and cytokine profile analysis. Research on the role of T cells in pemphigus has continued to progress, including that on T follicular helper cells, which initiate molecular mechanisms involved in antibody production in B cells. Autoreactive T cell research in mice has highlighted the crucial roles of cellular autoimmunity and improved the understanding of its pathogenesis, especially in paraneoplastic pemphigus. The mouse research has helped elucidate novel regulatory mechanisms of autoreactive T cells, such as thymic tolerance to desmoglein 3 and the essential roles of regulatory T cells, Langerhans cells, and other molecules in peripheral tissues. This review focuses on the immunological aspects of autoreactive T cells in pemphigus by providing detailed information on various related topics.

Decreased levels of T follicular helper (CD4+CXCR5+) cells and CD27+CD38+ and CD27+CD38− B cells in ankylosing spondylitis patients correlate with markers of inflammation

AbstractThe purpose of this study was to study CD4+CXCR5+ T follicular helper (TFH) cells, CD27+CD38+ plasmablasts and CD27+CD38− memory B cells, as well as disease‐related factors in patients with ankylosing spondylitis (AS) from northern Sweden. Peripheral blood mononuclear cells (PBMC) from 50 patients with AS (mean age 52 ± 9 years, 66% men, 100% HLA‐B27 positive) and 50 pairwise matched blood donor controls (mean age 54 ± 9 years, 66% men) were stained with antibodies for CD27, CD38, CD19, CD3, CD4 and CXCR5 markers and analysed by flow cytometry. Patients with AS were examined with spinal x‐ray for radiographic alterations (mSASSS), and plasma levels of C‐reactive protein, erythrocyte sedimentation rate, as well as selected proinflammatory and regulatory cytokines were determined. Physical mobility, function and disease activity were registered by BASMI, BASFI and ASDAS‐CRP, BASDAI, respectively. Comparing AS patients and controls pairwise, we observed a 56% reduction of TFH cells in PBMCs from AS patients (P = .000008). Furthermore, a 20%‐30% reduction in plasmablasts and B memory cells (P ≤ .002 and P ≤ .007, respectively) was observed. In female patients, negative correlations between ESR and TFH, plasmablasts and B memory cells were observed; Rs = −0.551, P ≤ .02; Rs = −0.476, P ≤ .05 and Rs = −0.522, P ≤ .03, respectively. In addition, positive correlations between the regulatory cytokine IL‐10 and the proportion of B cells, IL‐22, and the proportion of plasmablasts as well as a negative correlation between levels of the proinflammatory cytokine IL‐6 and TFH were detected. Our observations indicate a role of an aberrant humoral immune response related to inflammation in AS.