CD3 Cross-linking Research Articles

Human serum albumin-based drug-free macromolecular therapeutics induce apoptosis in chronic lymphocytic leukemia patient cells by crosslinking of CD20 and/or CD38 receptors.

This study explores the efficacy of human serum albumin (HSA)-based Drug-Free Macromolecular Therapeutics (DFMT) in treating Chronic Lymphocytic Leukemia (CLL), a prevalent adult leukemia subtype. DFMT, a novel strategy, employs biomimetic crosslinking of CD20 and CD38 receptors on malignant B cells without the need for low molecular weight drugs. Apoptosis is initiated via a two-step process: i) Recognition of a bispecific engager, Fab' fragment conjugated with morpholino oligonucleotide (Fab'-MORF1), by a cell surface antigen; followed by ii) crosslinking of the MORF1-decorated cells with a multivalent effector, HSA holding multiple copies of complementary MORF2, HSA-(MORF2)x. Herein we evaluated the efficacy of HSA-based DFMT in the treatment of 56 samples isolated from patients diagnosed with CLL. Fab' fragments from Obinutuzumab (OBN) and Isatuximab (ISA) were employed in the synthesis of anti-CD20 (Fab'OBN-MORF1) and anti-CD38 (Fab'ISA-MORF1) bispecific engagers. The efficacy of DFMT was significantly influenced by the expression levels of CD20 and CD38 receptors. Dual-targeting DFMT strategies (CD20 + CD38) were more effective than single-target approaches, particularly in samples with elevated receptor expression. Pretreatment of patient cells with gemcitabine or ricolinostat markedly increased cell surface CD20 and CD38 expression, respectively. Apoptosis was effectively initiated in 62.5% of CD20-targeted samples and in 42.9% of CD38-targeted samples. Our findings demonstrate DFMT's potential in personalized CLL therapy. Further research is needed to validate these outcomes in a larger number of patient samples and to explore DFMT's applicability to other malignancies.

Targeting NKG2D ligands in glioblastoma with a bispecific T-cell engager is augmented with conventional therapy and enhances oncolytic virotherapy of glioma stem-like cells

BackgroundGlioblastoma (GBM) almost invariably becomes resistant towards conventional treatment of radiotherapy and temozolomide (TMZ) chemotherapy, partly due to subpopulations of intrinsically resistant glioma stem-like cells (GSC). The oncolytic herpes simplex...

Engineering CD3/CD137 Dual Specificity into a DLL3-Targeted T-Cell Engager Enhances T-Cell Infiltration and Efficacy against Small-Cell Lung Cancer.

Small-cell lung cancer (SCLC) is an aggressive cancer for which immune checkpoint inhibitors (ICI) have had only limited success. Bispecific T-cell engagers are promising therapeutic alternatives for ICI-resistant tumors, but not all patients with SCLC are responsive. Herein, to integrate CD137 costimulatory function into a T-cell engager format and thereby augment therapeutic efficacy, we generated a CD3/CD137 dual-specific Fab and engineered a DLL3-targeted trispecific antibody (DLL3 trispecific). The CD3/CD137 dual-specific Fab was generated to competitively bind to CD3 and CD137 to prevent DLL3-independent cross-linking of CD3 and CD137, which could lead to systemic T-cell activation. We demonstrated that DLL3 trispecific induced better tumor growth control and a marked increase in the number of intratumoral T cells compared with a conventional DLL3-targeted bispecific T-cell engager. These findings suggest that DLL3 trispecific can exert potent efficacy by inducing concurrent CD137 costimulation and provide a promising therapeutic option for SCLC.

Abstract 6712: LBL-043, a novel LILRB4xCD3 T cell engager, for the treatment of LILRB4 positive leukemia

Abstract Background: Acute myeloid leukemia (AML) is a malignant clonal disorder linked to a broad spectrum of molecular alterations. It is still an incurable cancer, the prognosis is even worse in elderly patients, where overall survival (OS) at 1 year is approximately 10-15%. Now, several CD3 bispecific immunotherapies that harness T cells against AML are at various stages of preclinical and clinical development, but most of them are hampered by the lack of specific targets to distinguish tumor cells from normal hematopoietic stem cells (HSC). Leukocyte immunoglobulin-like receptor B4 (LILRB4) is an immune checkpoint inhibitory receptor, which is highly expressed on FAB M4 and M5 AML cells with a lack of expression on normal HSC and progenitor cells. LILRB4 expression level inversely correlates with OS of patients with M4 and M5 AML. All of these indicate that LILRB4 represents a promising target for developing a T cell redirecting bispecific antibody to treat patients with AML, especially for more aggressive M4 and M5 subtype. Herein a novel bispecific antibody targeting LILRB4 and CD3, LBL-043, is developed to specifically kill LILRB4 positive tumor cells by engaging T cells. Methods: LBL-043 was constructed using our proprietary LeadsBodyTM T cell engager platform, which is a 2:1 format bispecific antibody with two VHH arms targeting LILRB4 with high affinity and one scFv arm targeting CD3 with fine-tuned low affinity. The binding affinity of LBL-043 to LILRB4 and CD3 was determined with Fortebio, while the activity was measured using several cell based assays including reporter gene and TDCC assays. The anti-tumor activity of LBL-043 was investigated in B-NDG/hu-PBMC reconstructed mice implanted with MOLM-13 (human AML-M5a) tumor model. Results: The binding affinity of LBL-043 to LILRB4 and CD3 protein was 0.724 nM and 35.7 nM, respectively. In CD3 reporter gene assays, LBL-043 could activate the NFAT reporter signaling through LILRB4 binding dependent CD3 cross-linking with an EC50 value of 2.459 nM. LBL-043 was shown a potent TDCC activity in different LILRB4 expression level MOLM-13 cells and HL-60 cells with T cell activation and cytokine release. In B-NDG/hu-PBMC reconstructed mice implanted with MOLM-13 tumor cells, LBL-043 was shown significant anti-tumor activity. Simultaneously, LBL-043 can effectively improve the survival rate and maintain relatively stable body weight in the study compared to the vehicle group. Conclusion: LBL-043, a novel bispecific antibody targeting CD3 and LILRB4 with affinity differentiation, induces T cell killing of AML cells by simultaneously binding LILRB4 expressed on tumor cells and CD3 on T cells, redirecting T cells to efficiently killing of AML cells. It is also shown a great anti-tumor efficacy in CDX mouse models. These data support LBL-043 as a novel therapeutic bispecific antibody for the treatment of LILRB4 positive AML and CMML leukemia patients. Citation Format: Yujia Dang, Xiao Huang, Yurong Qin, Xiaoya Liu, Min Chen, Duqing Jiang, Guojin Wu, Mi Ye, Jianming Sun, Baohui Wang, Jing Guan, Tingting Li, Jordan Zhu, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-043, a novel LILRB4xCD3 T cell engager, for the treatment of LILRB4 positive leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6712.

ULCERATIVE COLITIS-DERIVED INTESTINAL EPITHELIAL CELLS ARE PROTECTED FROM CELL DEATH BY THE IMMUNE MEDIATORS RELEASED FROM RESTING AND ACTIVATED SYSTEMIC T CELLS

Abstract Immune dysregulation is a major driver in the pathogenesis of ulcerative colitis (UC), but the specific mechanisms of disease initiated by the adaptive immune system remain unknown. While many studies have focused on the contribution of individual T cell subsets, disrupted communication between the epithelium and T cells in the underlying lamina propria is not fully understood in UC. This study examines the impact of T cells isolated from healthy participants on primary cultures of epithelial cells derived from UC donors. Blood derived human CD4+ and CD8+ T cells were isolated by immunomagnetic negative selection and activated by anti-CD3/CD28 cross-linking. Activated and resting T cells were then co-cultured with UC-derived colonic epithelial spheroids, consisting predominantly of stem cells, suspended in Matrigel. Spheroid damage and epithelial cell death were measured by histological scoring of brightfield microscopic images and zombie violet positive staining, detected using flow cytometry, respectively. Our earlier studies showed that activated CD4+ T cells co-cultured with spheroids from healthy donors stimulated significant spheroid damage and epithelial cell death in a dose- and time-dependent manner. In addition, we reported that the addition of conditioned media (CM) from activated CD4+ T cells to healthy spheroids also induces spheroid damage and epithelial cell death. Neither resting CD4+ nor CD8+ T cells nor activated CD8+ T cells stimulate the same response (Panel A). Our preliminary results indicate that UC-derived epithelial cells, isolated from spheroids, exhibit a higher base line of spontaneous lethal sectoring (42%), when compared to spheroids isolated from healthy colons (10%). In stark contrast to healthy spheroids, addition of CM from resting and activated CD4+ or CD8+ T cells protect the ulcerative colitis-derived spheroids from damage and isolated UC epithelial cells from spontaneous death. We have not yet identified which T cell subset are protecting UC-derived epithelial cells from damage and death. Thus, our results distinguish the healthy epithelial spheroid response to T cell CM from UC spheroids in three distinct ways: [1] CM is protective in UC, [2] Both CD4 and CD8 cells mediate this response in UC, and [3] CM from resting and activated T cells secrete the responsible immune mediators. Overall, these results signify that the biological response of UC-derived epithelial cells to immune mediators is different from that seen with healthy epithelial cells, indicating a metabolic and genetic difference in the epithelium from the UC patient.

Englumafusp Alfa (CD19-4-1BBL) and Glofitamab Combination in Patients with Relapsed/Refractory Non-Hodgkin Lymphoma (R/R NHL): Biomarker Results from a Phase I Dose-Escalation Trial

Background: Englumafusp alfa is an antibody-like fusion protein that simultaneously targets CD19 on B cells and 4-1BB on T cells and other immune cells. In the presence of a T-cell receptor signal and strictly dependent on CD19 crosslinking, englumafusp alfa co-stimulates T cells via 4-1BB agonism that boosts T-cell effector functions and prevents T-cell anergy. An ongoing Phase I dose-escalation study (NCT04077723) is investigating the safety, efficacy, pharmacokinetics, and pharmacodynamics (PD) of intravenous administration of englumafusp alfa in combination with glofitamab in patients (pts) with R/R NHL. We recently presented preliminary clinical data demonstrating promising clinical activity and good tolerability in R/R NHL (Hutchings et al, ICML 2023). Here, we report preliminary peripheral blood (PB) and tissue biomarker analyses to demonstrate mechanisms of action (MoA), dose relationship, and baseline features associated with response. Methods: Exploratory biomarker analyses included data from 84 pts with indolent or aggressive NHL (aNHL) dosed with 0.36-75mg englumafusp alfa, starting on Cycle 2 Day 8 (C2D8) and followed by combination with the fixed target dose of glofitamab (30mg), once every 3 weeks, from C3D1 for up to 12 cycles. Immune profiling of PB by flow cytometry and plasma cytokines analysis by ELLA were performed. Circulating tumor DNA (ctDNA) dynamics were evaluated by the Avenio NHL CAPP-Seq assay (Stokowski et al, ASH 2022) at C3D1, C5D1, and the end of 12-cycle treatment (EoT) in patients with aNHL. Baseline tumor biopsies (n=65) were analyzed by immunohistochemistry/immunofluorescence assays. On-treatment (OT) PD changes during the first five cycles of treatment were evaluated and compared to historical glofitamab monotherapy PD data generated in a phase I/II study (NCT03075696). Results: Englumafusp alfa in combination with glofitamab induced PD changes in PB at all tested doses. Specifically, in a dose-dependent manner, englumafusp alfa in combination with glofitamab limited the PB expansion of fully differentiated PD1+ CD8+ effector memory T cells re-expressing CD45RA (Temra), previously shown to expand dose-dependently following glofitamab monotherapy (Broeske et al, Blood Advances 2022). Additionally, the combination also resulted in a significant PB expansion of the activated (HLA-DR+) and effector memory CD8+ T (T EM) cells, and the ratio of T EM to central memory and naïve CD8+ T cells compared to glofitamab monotherapy. Furthermore, we detected no significantly added cytokine release with the combination to that previously observed with glofitamab monotherapy, in line with the clinically observed safety profile. Preliminary analysis of ctDNA dynamics showed that at C3D1 33% (18/55), at C5D1 40% (15/38), and at EoT 53% (9/17) of the pts had no detectable ctDNA. Notably, 61% of pts with aNHL who achieved complete metabolic response (CMR) had no detectable ctDNA at any OT visit. Our preliminary analysis also indicated a significant (p=0.03) association between the observed OT expansion of CD8+ T EM cells and the extent of decrease in ctDNA levels (mean mutant molecules per milliliter) at EoT relative to baseline. Finally, we observed ~60% of pts with low tumor infiltrating CD8+ T cells (<500 cells/mm 2 CD8+) and a more aggressive disease (>5000 cells/mm 2 proliferative (Ki67+) tumor cells) at baseline, both poor outcome correlates of glofitamab monotherapy, achieved a CMR with higher doses of englumafusp alfa. Conclusions: In this study, we demonstrated the MoA of englumafusp alfa in R/R NHL and key PD effects in combination versus glofitamab monotherapy that will support optimal biological dose finding. Furthermore, the observed association between expansion of T EM cells in PB and changes in ctDNA dynamics at EoT, albeit preliminary, supports ctDNA as a marker for depth and durability of response. Overall, our PD and biomarker observations so far strengthen the rationale of combining glofitamab with englumafusp alfa to drive long-term responses in this heavily pre-treated NHL patient population. Acknowledgments: This study was sponsored by F. Hoffmann-La Roche Ltd. The authors would like to thank the patients who participated in this trial, their families, and their caregivers. Special thanks also to Daria Rukina, Iva Lelios, Philip Knuckles, Kat Reyskens, Grigori Singovski, and Joanne Hayward for their contributions to this work.

XFab-α4-1BB/CD40L fusion protein activates dendritic cells, improves expansion of antigen-specific T cells, and exhibits antitumour efficacy in multiple solid tumour models.

Additional immunotherapies are still warranted for non-responders to checkpoint inhibitors with refractory or relapsing cancers, especially for patients with "cold" tumours lacking significant immune infiltration at treatment onset. We developed XFab-α4-1BB/CD40L, a bispecific antibody targeting 4-1BB and CD40 for dendritic cell activation and priming of tumour-reactive T cells to inhibit tumours. XFab-α4-1BB/CD40L was developed by engineering an anti-4-1BB Fab arm into a CD40L trimer based on XFab® platform. Characterisation of the bispecific antibody was performed by cell-based reporter assays, maturation of dendritic cell assays, and mixed lymphocyte reactions. The abilities of antigen-specific T-cell expansion and antitumour efficacy were assessed in syngeneic mouse tumour models. Toxicological and pharmacodynamic profiles were investigated in non-human primates. XFab-α4-1BB/CD40L demonstrated independent CD40 agonistic activity and conditional 4-1BB activity mediated by CD40 crosslinking, leading to dendritic cell maturation and T-cell proliferation in vitro. We confirmed the expansion of antigen-specific T cells in the vaccination model and potent tumour regression induced by the bispecific antibody alone or in combination with gemcitabine in vivo, concomitant with improved tumour-reactive T-cell infiltration. XFab-α4-1BB/CD40L showed no signs of liver toxicity at doses up to 51mg/kg in a repeated-dose regimen in non-human primates. XFab-α4-1BB/CD40L is capable of enhancing antitumour immunity by modulating dendritic cell and T-cell functions via targeting 4-1BB agonism to areas of CD40 expression. The focused, potent, and safe immune response induced by the bispecific antibody supports further clinical investigations for the treatment of solid tumours.

IFN-γ is Necessary but not Sufficient to mediate Activated CD4+ T cell-induced epithelial cell damage and death

Abstract Bidirectional communication between the intestinal epithelial barrier and the immune cells in the underlying lamina propria is critical to human health. This study investigates the impact of human T cell subtypes on intestinal epithelial cell phenotype and survival. Blood-derived CD4+ or CD8+ T cells were isolated from healthy human participants using negative magnetic separation and co-cultured with healthy human colonic epithelial stem cell spheroids suspended in Matrigel. A subset of T cells was activated by anti-CD3/CD28 cross-linking for 24 h prior to co-culture. Only activated CD4+ T cells (ACD4) caused significant epithelial cell damage and death as measured by histological scoring of brightfield microscopic images and zombie violet+ epithelial cells detected by flow cytometry. The mechanism of cell damage and death was determined to be via soluble mediators, in that conditioned media from ACD4 but not activated CD8+ T cells (ACD8) significantly induced spheroid cell damage and death. A Luminex cytokine multiplex assay revealed that high levels of IFN-γ, IL-17, and TNF-α were produced by ACD4 as compared with ACD8. Neutralizing IFN-γ antibody completely protected spheroids from death induced by ACD4. In contrast, the addition of only IFN-γ protein did not induce spheroid cell death. This finding conflicts with currently published literature that indicates IFN-γ protein is able to induce spheroid cell death for both humans and mice. In addition, ACD8 also secrete IFN-γ, although to a lesser extent than ACD4, yet do not initiate spheroid cell death. These results indicate that there is a unique mixture of immune mediator released by ACD4s that mediate spheroid damage and death. DP1 DA-037997 T32 GM007250

Abstract 2965: LBL-033, a novel bispecific antibody targeting MUC16 and CD3, for the treatment of tumors over-expressing MUC16

Abstract Background: MUC16 is a large glycoprotein overexpressed on cell membrane of solid tumors, especially in almost 80% of ovarian cancer. The extracellular part of MUC16 could be cleaved as CA125, which is well known as a diagnosis biomarker for ovarian cancer. Despite recent advances in therapies such as PARP inhibitors, advanced ovarian cancer remains a disease of high unmet need. Herein a novel bispecific antibody targeting MUC16 and CD3, LBL-033, is developed to specifically kill MUC16 positive tumor cells by engaging T cells. Methods: LBL-033 was constructed using our unique T cell engager platform, which is a 2:1 format bispecific antibody with two arms targeting MUC16 membrane-proximal domain with high affinity and one arm targeting CD3 with fine-tuned affinity. The binding affinity of LBL-033 to MUC16 and CD3 was determined with Fortebio, while the activity was measured using several cell based assays including reporter gene and TDCC assays. The anti-tumor activity of LBL-033 was investigated in C57BL/6-huCD3e mice implanted with MC38-huMUC16 cells. GLP-compliant repeat-dose toxicologic study was evaluated in cynomolgus monkeys. Results: The binding affinity of LBL-033 to MUC16 and CD3 protein was 2.67 nM and 29.6 nM, respectively. The epitope that anti-MUC16 binds was identified at the C-terminal of MUC16 extracellular domain; and the activity was not affected in the presence of soluble CA-125. In CD3 reporter gene assay, LBL-033 could activate the NFAT reporter element through MUC16 antigen-dependent CD3 cross-linking with an EC50 value of 1.068 nM. LBL-033 was shown a potent TDCC activity in OVCAR3 cells with T cell activation. In C57BL/6-huCD3e transgenic mice implanted with MC38-huMUC16 tumor cells, LBL-033 was shown a dose dependent anti-tumor activity. In addition, LBL-033 had a significant synergistic effect, when combined with anti-PD-1. LBL-033 was shown favorable pharmacokinetic properties with a good safety profile in cynomolgus monkeys. Conclusion: LBL-033, a novel bispecific antibody targeting CD3 and MUC16 with a unique epitope, which is located at the MUC16 membrane-proximal domain, with affinity differentiation between anti-MUC16 and anti-CD3. It is shown a great anti-tumor efficacy in animal models, with a good safety profile in monkeys. These data support LBL-033 as a novel therapeutic bispecific antibody for ovarian cancer and other MUC16 positive tumors. A dose escalation study in humans will begin in Q1 2023. Citation Format: Jianming Sun, Xiao Huang, Yurong Qin, XingXing Fang, Min Chen, Huan Lin, Jing Guan, Jing Huang, Tongjun Liu, Baohui Wang, Guojin Wu, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-033, a novel bispecific antibody targeting MUC16 and CD3, for the treatment of tumors over-expressing MUC16 [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 2965.

Glutamine Metabolism Underlies the Functional Similarity of T Cells between Nile Tilapia and Tetrapod.

As the lowest organisms possessing T cells, fish are instrumental for understanding T cell evolution and immune defense in early vertebrates. This study established in Nile tilapia models suggests that T cells play a critical role in resisting Edwardsiella piscicida infection via cytotoxicity and are essential for IgM+ B cell response. CD3 and CD28 monoclonal antibody crosslinking reveals that full activation of tilapia T cells requires the first and secondary signals, while Ca2+ -NFAT, MAPK/ERK, NF-κB, and mTORC1 pathways and IgM+ B cells collectively regulate T cell activation. Thus, despite the large evolutionary distance, tilapia and mammals such as mice and humans exhibit similar T cell functions. Furthermore, it is speculated that transcriptional networks and metabolic reprogramming, especially c-Myc-mediated glutamine metabolism triggered by mTORC1 and MAPK/ERK pathways, underlie the functional similarity of T cells between tilapia and mammals. Notably, tilapia, frogs, chickens, and mice utilize the same mechanisms to facilitate glutaminolysis-regulated T cell responses, and restoration of the glutaminolysis pathway using tilapia components rescues the immunodeficiency of human Jurkat T cells. Thus, this study provides a comprehensive picture of T cell immunity in tilapia, sheds novel perspectives for understanding T cell evolution, and offers potential avenues for intervening in human immunodeficiency.

CD19 4-1BBL (RO7227166) a Novel Costimulatory Bispecific Antibody Can be Safely Combined with the T-Cell-Engaging Bispecific Antibody Glofitamab in Relapsed or Refractory B-Cell Non-Hodgkin Lymphoma

Background RO7227166 is a bispecific monoclonal antibody that simultaneously targets CD19 on B-cells and 4-1BB on T-cells and other immune cells. In the presence of a T-cell receptor signal and strictly dependent on CD19 crosslinking, RO7227166 provides a strong co-stimulation to T-cells via 4-1BB agonism. RO7227166 is therefore not exhibiting single-agent activity. Our preclinical and clinical data demonstrated 4-1BB upregulation on immune cells by glofitamab and in our in vivo humanized lymphoma xenograft mouse models the combination of glofitamab plus RO7227166 showed increased anti-tumor efficacy mediated by increased infiltration of activated CD8 T-cells within the tumor. We therefore hypothesized that RO7227166 would boost the anti-tumor activity of glofitamab in patients (pts) with B-cell non-Hodgkin lymphoma (B-NHL). Methods We report data from the first-in-human study BP41072 (NCT04077723), a multicenter open label Phase 1 trial investigating the safety, tolerability, pharmacokinetics (PK), pharmacodynamics and preliminary antitumor activity of RO7227166 in combination with glofitamab (part II of the study) in relapsed/refractory (R/R) B-NHL pts. Pts received 1000mg obinutuzumab 7 days prior to first glofitamab administration to mitigate cytokine release syndrome (CRS). Glofitamab was then dosed following a step-up regimen in cycle 1 (C1), from cycle 2 day 1 (C2D1) on, every 3 weeks (Q3W) for up to 12 cycles. First RO7227166 dose was given on C2D8 and from C3 onwards on the same day as glofitamab. RO7227166 dose escalation was guided by an mCRM EWOC model with overdose control. Response rates were reported based on the Lugano criteria. Results As of June 7, 2022, a total of 46 pts with R/R aggressive B-NHL (diffuse-large B-cell lymphoma [DLBCL]) and 24 pts with R/R indolent B-NHL (23 follicular lymphoma [FL], 1 marginal zone lymphoma [MZL]) received RO7227166 doses ranging from 360µg up to 33mg. So far the dose level with the expected maximum efficacy is not yet reached. Pts had a median age of 66 years, 44.3% were female, with ECOG of 0 (62.9%) or 1 (37.1%). Approximately half of pts had Stage IV disease (44.3%), 18.6% pts had bulky disease (at least one lesion > 6cm), 27.1% had prior CAR-T therapy. Median prior lines of therapy was 3 (range 1-7), with 40% refractory to last prior therapy, 21.4% primary refractory and 12.9% refractory to CAR-T. Across 70 safety evaluable pts, 67 (95.7%) had adverse events (84.3% pts grade 1, 64.3% pts grade 2, 44.3% pts grade 3, 8.6% pts grade 4 and 4.3% pts grade 5). Grade 5 events were pneumocystis jirovecii pneumonia which qualified as dose-limiting toxicity (n=1) and septic shock (n=1) and COVID-19 infection (n=1) which were both assessed as not related to study medications. The most frequent adverse event was CRS with 63 events occurring in 38 (54.3%) pts (maximum grade 1 in 37,1% pts, and maximum grade 2 in 17,1% pts, according to ASTCT score). Of all 63 events (including multiple events for some pts), 55 were related to glofitamab alone (87,3%), 5 events to glofitamab and RO7227166 (8%, all grade 1) and 3 events related to RO7227166 alone (4.8%, all grade 1). Overall the safety profile was mainly driven by glofitamab (Hutchings, et al. 2021) and we are yet to detect an additive safety signal from RO7227166 when combined with glofitamab. RO7227166 exposure increased dose-dependently across the investigated doses. Furthermore, preliminary pharmacodynamic analysis showed further expansion of primed and activated CD8, and effector memory T-cells in the periphery, which supports the expected mode of action of RO7227166 in combination with glofitamab. Of the 56 response-evaluated subjects, pts with DLBCL achieved a best overall response rate (BORR) of 67% and a complete response rate (CRR) of 39%, while the BORR and CRR for patients with FL was 91% and 73%. Summary This is the first study demonstrating that glofitamab can be safely combined with a costimulatory bispecific antibody (RO7227166) in R/R B-NHL. No new safety signals or signs of increased immune-related AEs were detected and the overall safety profile was consistent with that reported from single-agent glofitamab. At doses explored, combination therapy maintains efficacy similar to glofitamab single-agent, and dose escalation continues with additive benefit of the combination expected at higher doses.

Simultaneous crosslinking of CD20 and CD38 receptors by drug-free macromolecular therapeutics enhances B cell apoptosis in vitro and in vivo.

Drug-Free Macromolecular Therapeutics (DFMT) is a new paradigm in macromolecular therapeutics that induces apoptosis in target cells by crosslinking receptors without the need of low molecular weight drugs. Programmed cell death is initiated via a biomimetic receptor crosslinking strategy using a two-step approach: i) recognition of cell surface antigen by a morpholino oligonucleotide-modified antibody Fab' fragment (Fab'-MORF1), ii) followed by crosslinking with a multivalent effector motif - human serum albumin (HSA) grafted with multiple complementary morpholino oligonucleotides (HSA-(MORF2)x). This approach is effective in vitro, in vivo, and ex vivo on cells from patients diagnosed with various B cell malignancies. We have previously demonstrated DFMT can be applied to crosslink CD20 and CD38 receptors to successfully initiate apoptosis. Herein, we show simultaneous engagement, and subsequent crosslinking of both targets ("heteroreceptor crosslinking"), can further enhance the apoptosis induction capacity of this system. To accomplish this, we incubated Raji (CD20+; CD38+) cells simultaneously with anti-CD20 and anti-CD38 Fab'-MORF1 conjugates, followed by addition of the macromolecular crosslinker, HSA-(MORF2)x to co-cluster the bound receptors. Fab' fragments from Rituximab and Obinutuzumab were employed in the synthesis of anti-CD20 bispecific engagers (Fab'RTX-MORF1 and Fab'OBN-MORF1), whereas Fab' fragments from Daratumumab and Isatuximab (Fab'DARA-MORF1 and Fab'ISA-MORF1) targeted CD38. All heteroreceptor crosslinking DFMT combinations demonstrated potent apoptosis induction and exhibited synergistic effects as determined by Chou-Talalay combination index studies (CI<1). In vitro fluorescence resonance energy transfer (FRET) experiments confirmed the co-clustering of the two receptors on the cell surface in response to the combination treatment. The source of this synergistic therapeutic effect was further explored by evaluating the effect of combination DFMT on key apoptosis signaling events such as mitochondrial depolarization, caspase activation, lysosomal enlargement, and homotypic cell adhesion. Finally, a xenograft mouse model of CD20+/CD38+ Non Hodgkin lymphoma was employed to demonstrate in vivo the enhanced efficacy of the heteroreceptor-crosslinking DFMT design versus single-target systems.

Semimechanistic Physiologically-Based Pharmacokinetic/Pharmacodynamic Model Informing Epcoritamab Dose Selection for Patients With B-Cell Lymphomas.

Epcoritamab is a CD3xCD20 bispecific antibody (bsAb) that induces T-cell-mediated cytotoxicity against CD20-positive B cells. Target engagement and crosslinking of CD3 and CD20 (trimer formation) leads to activation and expansion of T cells and killing of malignant B cells. The primary objective of the dose-escalation part of the phase I/II trial of epcoritamab was to determine the maximum tolerated dose, recommended phase II dose (RP2D), or both. For bsAbs, high target saturation can negatively affect trimer formation. The unique properties and mechanisms of action of bsAbs require novel pharmacokinetic (PK) modeling methods to predict clinical activity and inform RP2D selection. Traditional PK/pharmacodynamic (PD) modeling approaches are inappropriate because they may not adequately predict exposure-response relationships. We developed a semimechanistic, physiologically-based PK/PD model to quantitatively describe biodistribution, trimer formation, and tumor response using preclinical, clinical PK, biomarker, tumor, and response data from the dose-escalation part of the phase I/II trial. Clinical trial simulations were performed to predict trimer formation and tumor response in patients with diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma (FL). Model-predicted trimer formation plateaued at doses of 48 to 96 mg. Simulation results suggest that the 48-mg dose may achieve optimal response rates in DLBCL and FL. Exposure-safety analyses showed a flat relationship between epcoritamab exposure and risk of cytokine release syndrome in the dose range evaluated. This novel PK/PD modeling approach guided selection of 48 mg as the RP2D and provides a framework that may be applied to other CD3 bsAbs.

Type of PaperY192 within the SH2 Domain of Lck Regulates TCR Signaling Downstream of PLC-γ1 and Thymic Selection.

Signaling via the TCR, which is initiated by the Src-family tyrosine kinase Lck, is crucial for the determination of cell fates in the thymus. Because of its pivotal role, ablation of Lck results in a profound block of T-cell development. Here, we show that, in addition to its well-known function in the initiation of TCR signaling, Lck also acts at a more downstream level. This novel function of Lck is determined by the tyrosine residue (Y192) located in its SH2 domain. Thymocytes from knock-in mice expressing a phosphomimetic Y192E mutant of Lck initiate TCR signaling upon CD3 cross-linking up to the level of PLC-γ1 phosphorylation. However, the activation of downstream pathways including Ca2+ influx and phosphorylation of Erk1/2 are impaired. Accordingly, positive and negative selections are blocked in LckY192E knock-in mice. Collectively, our data indicate that Lck has a novel function downstream of PLCγ-1 in the regulation of thymocyte differentiation and selection.

Abstract 5554: A bispecific T cell engager targeting CEACAM5/6 exhibits strong anti-tumor efficacy in pre-clinical studies

Abstract Background: The carcinoembryonic antigen-related adhesion molecules (CEACAM), containing seven members, are involved in many biological processes including cell adhesion, proliferation, differentiation, and tumor suppression. Some variants of CEACAMs such as CEACAM5 and CEACAM6 have long been recognized to be overexpressed in many cancers and highly associated with cancer development. Immunotherapies targeting CEACAM have achieved some clinical benefit, e.g. Tusamitamab Ravtansine (anti-CEACAM5 ADC), that has shown decent disease control in NSCLC. Recently, with the success of CAR-T and T cell engager (TCE) therapies against blood cancers, TCEs against solid tumors have also been developed. For instance, Cibisatamab, targeting CEACAM5 and CD3, is currently under clinical investigation in CRC patients. Here, we report the discovery of a bispecific antibody (biAb) anti-CEACAM5/6 x CD3 that mediates direct T cell killing against CEACAM5- and/or CEACAM6-expressing cancer cells. Methods: Anti-CEACAM5/6 x CD3 TCE consists of two CEACAM5/6 binding Fabs for the preferential recognition of CEACAM5/6-high-expressing cancer cells, and one CD3 binding domain that permits CEACAM5/6-mediated CD3 crosslinking and T cell activation. To reduce off-target T cell stimulation there is no CH2 in this format to eliminate Fc receptor binding, while an anti-HSA VHH was included into this construct for half-life extension. Binding affinity and specificity were studied by flow cytometry, IHC and on the Retrogenix Cell Microarray Technology platform, a human membrane/secreted protein binding array. The immunomodulatory functions were evaluated using luciferase reporter cell assays, cell killing assays in vitro and via PBMC-based Hu-NSG mouse models in vivo. Results: Our CEACAM5/6 binding antibody only interacted with CEACAM5 and CEACAM6 on the Retrogenix Cell Microarray against over 5,500 proteins. Importantly, it showed a similar sensitivity to recognize CRC tumor tissues in an IHC array as the benchmark (BMK), but significantly better selectivity at recognition of tumor tissues vs. normal tissues. Next, in vitro testing illustrated CEACAM5/6-mediated T cell activation by the TCE, and such stimulation also correlated with the binding affinity of the anti-CD3 domain. The lead candidate with the optimized CD3 binding affinity, showed better anti-tumor efficacy than the BMK in mice. Furthermore, this molecule displayed a typical PK as a conventional antibody with a mean T1/2 of around 115hrs. Finally, we show high tolerance in NHP of which no obvious signs of toxicity were observed at 2.5 mg/kg. Conclusion: In summary, a bispecific TCE with high selectivity to CEACAM5/6 overexpressing cancer cells was engineered. It displays potent anti-tumor efficacy, and support from target selectivity testing shows promising safety traits. Citation Format: Liandi Chen, Weifeng Huang, Xiaoniu Miao, Jiayi Si, Chao Wang, Tiantian Dong, Andy Tsun, Yi Luo. A bispecific T cell engager targeting CEACAM5/6 exhibits strong anti-tumor efficacy in pre-clinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5554.

Phase I study of intravesical anti-CD40 agonist antibody 2141-V11 for non-muscle invasive bladder cancer unresponsive to Bacillus Calmette-Guerin (BCG) therapy.

TPS4616 Background: CD40 is an immune-stimulatory receptor centrally involved in activating antigen-presenting cells and downstream anti-tumor immunity. Multiple agonistic anti-CD40 antibodies have been investigated clinically for cancer therapy, but have thus far shown limited efficacy and notable systemic toxicity. Our group has previously demonstrated that the ability of the Fc domain of anti-CD40 agonist antibodies to engage the FcγRIIB receptor, needed for optimal CD40 crosslinking, is critical for CD40 agonist activity. We engineered a fully-human anti-CD40 agonist antibody (2141-V11) with optimized Fc binding to FcγRIIB, which demonstrates significantly enhanced CD40 agonist activity. Non-muscle invasive bladder cancer (NMIBC), which remains an area of significant unmet need, represents an attractive clinical context to pursue CD40 agonist therapy given strong enrichment of CD40 in the bladder tumor microenvironment and routine use of local intravesical delivery as a potential strategy to mitigate systemic toxicity. Our preclinical studies, including in immunocompetent orthotopic murine models of bladder cancer humanized for CD40 and Fcγ receptors, revealed that intravesical delivery of 2141-V11 induced potent and durable anti-tumor immunity in both front-line and BCG-unresponsive settings without associated systemic toxicity (Garris and Wong, et al., Sci Transl Med, 2021;13:eabd1346). These data provide rationale to investigate intravesical 2141-V11 for the treatment of BCG-unresponsive NMIBC. Methods: This is a phase 1, open-label, dose-escalation study (NCT05126472) to evaluate the safety and tolerability of intravesically-delivered 2141-V11 in patients with BCG-unresponsive NMIBC who are considered ineligible for or have elected not to undergo radical cystectomy. Key inclusion criteria include adults with high-grade NMIBC (Ta, CIS, and/or T1) of urothelial histology that is unresponsive to adequate BCG therapy per consensus 2018 FDA guidelines. The primary objectives are to evaluate the safety and tolerability of 2141-V11 and to define the maximum tolerated dose and/or recommended phase 2 dose. The secondary objectives are to characterize the pharmacokinetics and evaluate the preliminary clinical activity of 2141-V11. Exploratory objectives include assessment of biomarkers of biological activity and disease resistance, their potential associations with clinical outcome measures, and patient-reported outcomes/quality of life measures. The 2141-V11 antibody is administered intravesically once weekly for 3 doses. Depending on disease status at week 13 and week 25 evaluations, patients may be eligible for re-treatment (once weekly for 3 doses) at these time points. Dose exploration utilizes a modified continual reassessment method design. Enrollment began in November 2021 and is ongoing. Clinical trial information: NCT05126472.

Acute Csk inhibition hinders B cell activation by constraining the PI3 kinase pathway

T cell antigen receptor (TCR) and B cell antigen receptor (BCR) signaling are initiated and tightly regulated by Src-family kinases (SFKs). SFKs positively regulate TCR signaling in naïve T cells but have both positive and negative regulatory roles in BCR signaling in naïve B cells. The proper regulation of their activities depends on the opposing actions of receptor tyrosine phosphatases CD45 and CD148 and the cytoplasmic tyrosine kinase C-terminal Src kinase Csk. Csk is a major negative regulator of SFKs. Using a PP1-analog-sensitive Csk (CskAS) system, we have previously shown that inhibition of CskAS increases SFK activity, leading to augmentation of responses to weak TCR stimuli in T cells. However, the effects of Csk inhibition in B cells were not known. In this study, we surprisingly found that inhibition of CskAS led to marked inhibition of BCR-stimulated cytoplasmic free calcium increase and Erk activation despite increased SFK activation in B cells, contrasting the effects observed in T cells. Further investigation revealed that acute CskAS inhibition suppressed BCR-mediated phosphatidylinositol 3,4,5-trisphosphate (PIP3) production in B cells. Restoring PIP3 levels in B cells by CD19 cross-linking or SHIP1 deficiency eliminated the negative regulatory effect of CskAS inhibition. This reveals the critical role of Csk in maintaining an appropriate level of SFK activity and regulating PIP3 amounts as a means of compensating for SFK fluctuations to prevent inappropriate B cell activation. This regulatory mechanism controlling PIP3 amounts may also contribute to B cell anergy and self-tolerance.

A reporter gene assay for determining the biological activity of therapeutic antibodies targeting TIGIT

T cell immunoglobulin and ITIM domain (TIGIT) is a novel immune checkpoint that has been considered as a target in cancer immunotherapy. Current available bioassays for measuring the biological activity of therapeutic antibodies targeting TIGIT are restricted to mechanistic investigations because donor primary T cells are highly variable. Here, we designed a reporter gene assay comprising two cell lines, namely, CHO-CD112-CD3 scFv, which stably expresses CD112 (PVRL2, nectin-2) and a membrane-bound anti-CD3 single-chain fragment variable (scFv) as the target cell, and Jurkat-NFAT-TIGIT, which stably expresses TIGIT as well as the nuclear factor of activated T-cells (NFAT) response element-controlled luciferase gene, as the effector cell. The anti-CD3 scFv situated on the target cells activates Jurkat-NFAT-TIGIT cells through binding and crosslinking CD3 molecules of the effector cell, whereas interactions between CD112 and TIGIT prevent activation. The presence of anti-TIGIT mAbs disrupts their interaction, which in turn reverses the inactivation and luciferase expression. Optimization and validation studies have demonstrated that this assay is superior in terms of specificity, accuracy, linearity, and precision. In summary, this reliable and effective reporter gene assay may potentially be utilized in lot release control, stability assays, screening, and development of novel TIGIT-targeted therapeutic antibodies.

CD40 Cross-Linking Induces Migration of Renal Tumor Cell through Nuclear Factor of Activated T Cells (NFAT) Activation.

CD40 crosslinking plays an important role in regulating cell migration, adhesion and proliferation in renal cell carcinoma (RCC). CD40/CD40L interaction on RCC cells activates different intracellular pathways but the molecular mechanisms leading to cell scattering are not yet clearly defined. Aim of our study was to investigate the main intracellular pathways activated by CD40 ligation and their specific involvement in RCC cell migration. CD40 ligation increased the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH (2)-terminal kinase (JNK) and p38 MAPK. Furthermore, CD40 crosslinking activated different transcriptional factors on RCC cell lines: AP-1, NFkB and some members of the Nuclear Factor of Activated T cells (NFAT) family. Interestingly, the specific inhibition of NFAT factors by cyclosporine A, completely blocked RCC cell motility induced by CD40 ligation. In tumor tissue, we observed a higher expression of NFAT factors and in particular an increased activation and nuclear migration of NFATc4 on RCC tumor tissues belonging to patients that developed metastases when compared to those who did not. Moreover, CD40-CD40L interaction induced a cytoskeleton reorganization and increased the expression of integrin β1 on RCC cell lines, and this effect was reversed by cyclosporine A and NFAT inhibition. These data suggest that CD40 ligation induces the activation of different intracellular signaling pathways, in particular the NFATs factors, that could represent a potential therapeutic target in the setting of patients with metastatic RCC.

FcεRIγ-negative NK cells persist in vivo and enhance efficacy of therapeutic monoclonal antibodies in multiple myeloma

AbstractMonoclonal antibodies (mAbs) are a central component of therapy for hematologic malignancies. Widely used mAb agents in multiple myeloma (MM) include daratumumab and elotuzumab. However, not all patients respond to these agents, and resistance is a significant clinical issue. A recently discovered subset of human natural killer (NK) cells lacking expression of FcεRIγ (g-NK cells) was found to have a multifold increase in antibody-dependent effector functions after CD16 crosslinking. In this study, we tested the capacity of g-NK cells to enhance the efficacy of therapeutic mAbs against MM. In vitro, we found that g-NK cells have strikingly superior anti-myeloma cytotoxicity compared with conventional NK (cNK) cells when combined with daratumumab or elotuzumab (∼sixfold; P < .001). In addition, g-NK cells naturally expressed minimal surface CD38 and SLAMF7, which reduced the incidence of therapeutic fratricide. In tumor-naïve murine models, the persistence of g-NK cells in blood and spleen was >10 times higher than that of cNK cells over 31 days (P < .001). In vivo efficacy studies showed that the combination of daratumumab and g-NK cells led to a >99.9% tumor reduction (by flow cytometry analysis) compared with the combination of daratumumab and cNK cells (P < .001). Moreover, treatment with daratumumab and g-NK cells led to complete elimination of myeloma burden in 5 of 7 mice. Collectively, these results underscore the unique ability of g-NK cells to potentiate the activity of therapeutic mAbs and overcome limitations of current off-the-shelf NK cell therapies without the need for cellular irradiation or genetic engineering.