Targeting CD20 Research Articles

Suppression of MUC1-Overexpressing Tumors by a Novel MUC1/CD3 Bispecific Antibody.

Mucin1 (MUC1) is abnormally glycosylated and overexpressed in a variety of epithelial cancers and plays a critical role in tumor progression. MUC1 has received remark attention as an oncogenic molecule and is considered a valuable tumor target for immunotherapy, while many monoclonal antibodies (mAbs) targeting MUC1-positive cancers in clinical studies lack satisfactory results. It would be highly desirable to develop an effective therapy against MUC1-expressing cancers. In this study, we constructed a novel T cell-engaging bispecific antibody (BsAb) targeting MUC1 and CD3 with the Fab-ScFv-IgG format. A high quality of MUC1-CD3 BsAb can be acquired through a standard method. Our study suggested that this BsAb could specifically bind to MUC1- and CD3-positive cells and efficiently enhance T cell activation, cytokine release, and cytotoxicity. Furthermore, our study demonstrated that this BsAb could potently redirect T cells to eliminate MUC1-expressing tumor cells in vitro and significantly suppress MUC1-positive tumor growth in a xenograft mouse model. Thus, T cell-engaging MUC1/CD3 BsAb could be an effective therapeutic approach to combat MUC1-positive tumors and our MUC1/CD3 BsAb could be a promising candidate in clinical applications for the treatment of MUC1-positive cancer patients.

Binding domain on CD22 molecules contributing to the biological activity of T cell-engaging bispecific antibodies

CD22, as the B-cell malignancies antigen, has been targeted for immunotherapies through CAR-T cells, antibody-drug conjugates (ADCs) and immunotoxins via interaction of antibodies with binding domains on the receptor. We hypothesized that avidity and binding domain of antibody to target cells may have significant impact on the biological function in tumor immunotherapy, and T cell-engaging bispecific antibody (TCB) targeting CD22 could be used in the therapy of hematologic malignancies. So, to address the question, we utilized the information of six previously reported CD22 mAbs to generate CD22-TCBs with different avidity to different domains on CD22 protein. We found that the avidity of CD22-TCBs to protein was not consistent with the avidity to target cells, indicating that TCBs had different binding mode to the protein and cells. In vitro results indicated that CD22-TCBs mediated cytotoxicity depended on the avidity of antibodies to target cells rather than to protein. Moreover, distal binding domain of the antigen contributed to the avidity and biological activity of IgG-[L]-scfv-like CD22-TCBs. The T cells' proliferation, activation, cytotoxicity as well as cytokine release were compared, and G5/44 BsAb was selected for further in vivo assessment in anti-tumor activity. In vivo results demonstrated that CD22-TCB (G5/44 BsAb) significantly inhibited the tumors growth in mice. All these data suggested that CD22-TCBs could be developed as a promising candidate for B-cell malignancies therapy through optimizing the design with avidity and binding domain to CD22 target in consideration.

Target-independent Immune-cell Activation by Aggregates of T Cell-redirecting Bispecific Antibodies

T cell-redirecting bispecific antibodies (bsAbs) have been under development as a new class of biotherapeutics for cancer immunotherapy. T cell-redirecting bsAbs simultaneously bind tumor-associated antigens on tumor cells and CD3 on T cells, resulting in T cell-mediated cytotoxicity against tumor cells. In this study, we prepared a tandem scFv-typed bsAb targeting HER2 and CD3 (HER2-CD3), and evaluated the impact of aggregation of HER2-CD3 on the in vitro immunotoxicity. A cell-based assay using CD3-expressing reporter cells revealed that the aggregates of HER2-CD3 directly activated CD3-expressing immune cells in the absence of target antigen (HER2)-expressing cells. Comparison of the aggregates generated under various stress conditions indicated the possibility that insoluble protein particles, which were detected by qLD analysis and contained non-denatured functional domains, contributed to the activation of CD3-expressing immune cells. In addition, HER2-CD3 aggregates stimulated hPBMCs and strongly induced the secretion of inflammatory cytokines and chemokines. The cytokine/chemokine-release profiles suggested that the aggregates could induce inflammatory responses not only by CD3-mediated T cell activation but also by other immune cell activations. These results indicated the potential risk of aggregation of T cell-redirecting bsAbs, which could induce unwanted immune cell activation and inflammation and thereby immune-mediated adverse reactions.

The Safety and Efficacy of Rituximab and Belimumab in Systemic Lupus Erythematosus: A Systematic Review.

There is a vital role of B cells in the pathogenesis of Systemic Lupus Erythematosus (SLE). Belimumab (Bel), an inhibitor of B cell activating factor (BAFF), and Rituximab (RTX), a monoclonal antibody targeting Cd20 antigen, have been used to manage systemic lupus. Several randomized controlled trials (RCTs) have evaluated these two agents' clinical efficacy and safety in different manifestations of SLE.This study aims to review the randomized control trials involving these two agents systematically and to explain if any disparity is noticed in the primary and secondary outcomes between these two agents. This study is done according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)guidelines. After applying the inclusion criteria and quality assessment by independent reviewers and co-authors, relevant papers were identified, and data were extracted. The results have shown thatRCTs involving Belimumab achieved primary endpoints; however, targeted endpoints were not achieved in studiesinvolving Rituximab. It is concluded that despite the conflicting results obtained in clinical trials, both are effective in systemic lupus, as indicated in real-world clinical experience. However, better-designed multicenter studies evaluating these B-cell-targeting drugs are needed.

Targeting CD137 (4-1BB) towards improved safety and efficacy for cancer immunotherapy.

T cells play a critical role in antitumor immunity, where T cell activation is regulated by both inhibitory and costimulatory receptor signaling that fine-tune T cell activity during different stages of T cell immune responses. Currently, cancer immunotherapy by targeting inhibitory receptors such as CTLA-4 and PD-1/L1, and their combination by antagonist antibodies, has been well established. However, developing agonist antibodies that target costimulatory receptors such as CD28 and CD137/4-1BB has faced considerable challenges, including highly publicized adverse events. Intracellular costimulatory domains of CD28 and/or CD137/4-1BB are essential for the clinical benefits of FDA-approved chimeric antigen receptor T cell (CAR-T) therapies. The major challenge is how to decouple efficacy from toxicity by systemic immune activation. This review focuses on anti-CD137 agonist monoclonal antibodies with different IgG isotypes in clinical development. It discusses CD137 biology in the context of anti-CD137 agonist drug discovery, including the binding epitope selected for anti-CD137 agonist antibody in competition or not with CD137 ligand (CD137L), the IgG isotype of antibodies selected with an impact on crosslinking by Fc gamma receptors, and the conditional activation of anti-CD137 antibodies for safe and potent engagement with CD137 in the tumor microenvironment (TME). We discuss and compare the potential mechanisms/effects of different CD137 targeting strategies and agents under development and how rational combinations could enhance antitumor activities without amplifying the toxicity of these agonist antibodies.

Five-year subgroup analysis of tafasitamab + lenalidomide from the phase II L-MIND study in patients with relapsed or refractory diffuse large B-cell lymphoma.

e19522 Background: The Phase II L-MIND study led to accelerated US approval and EU conditional authorization of the CD19-targeted immunotherapy, tafasitamab, + lenalidomide (LEN) followed by tafasitamab monotherapy for patients (pts) with R/R DLBCL ineligible for autologous stem cell transplant (ASCT). Long-term L-MIND data further support the regimen. Here we present exploratory analyses of final 5-year (yr) efficacy in subgroups of interest. Methods: Pts (≥18 years) with ASCT-ineligible R/R DLBCL, 1–3 prior systemic therapies (incl. ≥1 targeting CD20) and ECOG PS 0–2 received tafasitamab for ≤12 28-day cycles (+ LEN), then alone until disease progression. Primary endpoint was objective response rate (ORR). Secondary endpoints included duration of response (DoR), progression-free survival (PFS) and overall survival (OS). Exploratory subgroup analyses including by International Prognostic Index (IPI) and time to progression after 1L (in pts with only 1 prior line) used Kaplan-Meier estimates of 5-yr endpoints. Results: As of 14 Nov, 2022 in the full analysis set (FAS; n=80), ORR was 56.2% [95% CI: 44.7–67.3]. Median treatment duration was 9.0 months (mo) [0.5–73.6] and median follow-up (mFU) for OS was 65.6 mo [59.9–70.3]). Median DoR was not reached (mFU: 43.7 mo [29.9–58.4]). Of 18 patients in follow-up for ≥5 years, 9 received tafasitamab until end of study per protocol, 9 discontinued while in remission. RR and 5-yr rates for DoR, PFS and OS showed long-term clinical activity in all pt subgroups (Table). Conclusions: In this 5-yr subgroup analysis, a long-term clinical benefit with tafasitamab + LEN followed by tafasitamab monotherapy was observed in all subgroups of clinical interest, including pts with poor prognosis risk factors. These data suggest this immunotherapy may have curative potential, which is being explored in further studies. Clinical trial information: NCT02399085 . [Table: see text]

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Off-the-shelf allogeneic T-cell therapies face the major challenges of graft-versus-host disease (GVHD) and graft rejection mediated by host and recipient alloreactive T cells, respectively. To address GVHD, we are using Epstein-Barr Virus-specific T cells (EBVST), which are virus specific rather than allo-specific and have not produced GVHD in over 300 allogeneic recipients. To prevent graft rejection, we modified the EBVST with a chimeric antigen receptor (CAR) targeting CD30, an antigen that is upregulated on allo-activated T-cells, which will consequently become targets for CD30.CAR EBVST. Hence, CD30.CAR EBVST can kill both CD30+ lymphoma cells and alloreactive T cells through their CAR, and may be boosted in vivo in EBV+ recipients via their native TCR. We are evaluating this approach in a phase 1 trial (NCT04288726) in patients with CD30+ lymphoma. We generated a bank of 7 CD30.CAR EBVST products by stimulating PBMC from healthy donors with overlapping EBV peptide mixtures followed by retroviral transduction with a 2nd generation, CD28-containing CD30.CAR. We have treated 16 patients (median age 36, range 22–53) with relapsed or refractory HL. Patients had undergone a median of 5 (range 3–7) prior lines of treatment. Escalating doses of 4 × 107 − 8 × 108 (DL1–4) CD30.CAR EBVST were infused after lymphodepletion. The frequency of CD30.CAR EBVST was measured by real time qPCR for the transgene in blood. Changes in the frequency of T-cells responding to EBV or tumor-associated antigens (epitope spreading) were measured by IFNɣ ELISpots. Clinical responses were assessed per Lugano criteria by PET/CT scans performed 4–8 wks post infusion. CD30.CAR EBVST were well tolerated, with no GVHD and only two patients having reversible grade 4 cytopenia. Five patients at DL3–4 had grade 1 cytokine release syndrome (CRS), but all resolved without treatment. Of the 16 patients treated, 6 had partial responses (PR) and 6/13 patients at DL2–4 had complete responses (CR), giving an overall objective response rate (ORR) of 75%. Despite these remarkable responses, qPCR for the CD30.CAR transgene showed near background levels by wk 1 post infusion. Although this could mean that CAR-T are quickly eliminated by alloreactive T-cells, additional infusions from the same donor product into the same patient can be associated with response (Figure 1); an alloreactive response would be anticipated to be amplified after each infusion and preclude additional responses. We are therefore analyzing tumor biopsy samples to determine the presence of CD30.CAR EBVST at tumor sites and characterize their phenotype for functionality. The research was funded by: Leukemia and Lymphoma Society SCOR (Heslop) and Tessa Therapeutics (Rooney/Ramos) through a grant to Baylor College of Medicine. Keywords: cellular therapies, Hodgkin lymphoma Conflicts of interests pertinent to the abstract C. A. Ramos Consultant or advisory role: Novartis, Genentech, CRISPR Therapeutics Research funding: Tessa Therapeutics, Athenex Therapeutics Other remuneration: Royalties: Tessa Therapeutics D. H. Quach Research funding: Tessa Therapeutics P. D. Lulla Research funding: Marker Therapeutics, Allovir R. H. Rouce Consultant or advisory role: Pfizer, Novartis Honoraria: Novartis, Medline Plus Research funding: Tessa Therapeutics B. J. Grilley Consultant or advisory role: Allovir, Marker Therapeutics, Tessa Therapeutics, Lokon Pharma, March Bio and Proxima Clinical Research Stock ownership: Allovir, QBRegulatory LLC N. Lapteva Consultant or advisory role: Tessa Therapeutics Research funding: Tessa Therapeutics M. K. Brenner Employment or leadership position: Founder Member Marker Therapeutics Consultant or advisory role: Tessa Therapeutics, Marker Therapeutics, Allogene, Walking Fish, Athenex, Turnstone Biologics, Posedia, Tscan, Bluebird Bio, Adaptimmune Therapeutics, Adintus, Onkimure, Triumvira Immunologics, Bellicum Pharmaceuticals, Memgen LLC, Brooklyn Immunotherapeutics, Coya, Astrazeneca Pharmaceuticals Stock ownership: Allovir, Marker Therapeutics, Tessa Therapeutics, Turnstone, Bluebird Bio, Allogene, Walking Fish, AlloVir, Tessa, Tscan Other remuneration: Royalties: Takeda, Bellicum, Marker Therapeutics H. E. Heslop Consultant or advisory role: Gilead Biosciences, Novartis, Tessa Therapeutics, Marker Therapeutics, Kiadis, PACT Pharma, Mesoblast, Ankyra Therapeutics Stock ownership: Allovir, Marker Therapeutics, Fresh Wind Biotechnologies, CoRegen Inc. Research funding: Tessa Therapeutics, Athenex Therapeutics C. M. Rooney Consultant or advisory role: Tessa Therapeutics, Marker Therapeutics, Cell Genix Stock ownership: Allovir, Marker Therapeutics Research funding: Tessa Therapeutics Other remuneration: Royalties: Takeda, Marker, Allovir, Bellicum

Tarlatamab in small cell lung cancer (SCLC): Safety and efficacy analyzed by baseline brain metastasis.

8582 Background: Tarlatamab, a BiTE molecule targeting DLL3 and CD3, is being studied in patients (pts) with extensive stage small cell lung cancer (ES-SCLC).1 Up to 25% of pts diagnosed with SCLC have brain metastases (BM) and 50% or more will develop BM during the course of the disease. Pts with ES-SCLC and BM have poor prognosis and quality of life. We analyzed the characteristics and outcomes of pts included in the tarlatamab phase 1 study (NCT03319940) who had baseline BM vs those who did not. Methods: Pts included in the phase 1 trial had SCLC that progressed after ≥1 platinum-based regimen and received tarlatamab (0.003–100 mg IV). Pts with treated BM were eligible if local therapy was delivered ≥ 2 weeks prior to first tarlatamab dose. Results: As of 03 January 2023, 192 pts had received tarlatamab as monotherapy. Median age was 62 (range, 32–80) y; 188 pts (98%) had ECOG PS 0–1 and median prior therapy lines was 2 (range, 1–6), with 107 pts (56%) receiving prior anti-PD1/PD-L1. 48 pts (25%) had BM at screening and 142 (74%) did not (No BM); data was missing for 2 pts (1%). Pts with BM were more likely to have received > 3 prior lines of therapy (BM, 29%; No BM, 9%) and have a record of prior brain radiation (BM, 85%; No BM, 40%); otherwise, characteristics of pts with BM were generally similar to those without. Tarlatamab delivered comparable activity in pts with BM vs. those without in terms of objective response rate (ORR; 20%, 25%) and disease control rate (DCR; 59%, 50%) (Table). Median duration of response (DoR) was 14.9 months (BM) vs. 13.0 months (no BM). Median overall survival (OS) was 13.2 months in the BM group vs 15.5 months in the no BM group. Safety outcomes were similar in terms of treatment-related AEs (TRAEs; BM, 90%; No BM, 94%) and grade (gr) ≥ 3 (BM, 38%; No BM, 39%). Groups were also comparable with respect to immune effector cell-associated neurotoxicity syndrome (ICANS) and associated neurologic events (any-grade, 6% vs 9%; gr ≥ 3, 4% vs 4%) and neutropenia (any-grade, 10% vs 15%; gr ≥ 3, 8% vs 8%). AEs numerically different in the BM vs No BM group included gr ≥ 4 TRAEs (17%, 7%) and any-grade CRS (67%, 54%). The only grade 5 TRAE (pneumonitis) occurred in the BM group. Conclusions: Tarlatamab continues to show promising efficacy and is generally safe in pts with ES SCLC irrespective of the presence of BM. Rates of ICANS and associated neurologic AEs were comparable between those with and without BM at baseline. Reference: 1. Paz-Ares L, et al. J Clin Oncol. DOI:10.1200/JCO.22.02823. Clinical trial information: NCT03319940 . [Table: see text]

CAR-T cells targeting CD38 and LMP1 and anti-tumor activity against NK/T cell lymphoma.

e19529 Background: Natural killer/T-cell lymphoma (NKTCL) is an aggressive lymphoma with poor prognosis. In the past decade, chimeric antigen receptor transduced T (CAR-T) cell therapy has been a promising strategy against hematologic malignancies. Researchers have evaluated the efficacy of CAR-T cell targeting CD38 in multiple myeloma and NKTCL. LMP1 may induce a malignant transformation in B cells and epithelial cells, making it a non-negligible molecule in NKTCL. These suggest the potential clinical administration of CD38 and LMP1 targeting therapy. Methods: Four second-generation CAR-T cells including two single-targeted CAR (CD38-CAR and LMP1-CAR) and two tandem CAR (CD38-LMP1 Tan CAR 1 and Tan CAR 2) were constructed. Then the effects of four CAR-T cells on NKTCL cells were evaluated both in vitro and in vivo. Results: Four CAR-T cells could effectively eliminate malignant target-positive NKTCL cells in vitro. They could be activated and produce inflammatory cytokines in a target-dependent manner. Stimulated by CD38+LMP1+ NKTCL cells, two Tan CAR-T cells produced more IL-13, Granzyme B, IFN-γ and TNF-α than LMP1-CAR-T cells. In vivo test showed that CAR-T cells also exhibit significant anti-tumor effects in a xenotransplant NKTCL mice models. Two Tan CAR-T cells showed higher cytolytic activity against CD38+LMP1+ NKTCL cells than single-targeted CAR-T cells both in vitro and in vivo. Conclusions: We explored the feasibility of CAR-T cell therapy targeting both CD38 and LMP1 molecule. Our results indicated a promising effective therapeutic strategy for NKTCL.

A Novel bispecific T-cell engager (BiTE) targeting CD22 and CD3 has both in vitro and in vivo activity and synergizes with blinatumomab in an acute lymphoblastic leukemia (ALL) tumor model

Immunotherapy has revolutionized cancer therapy. Two recently FDA-approved immunotherapies for B-cell malignancies target CD19, in the form of a Bispecific T-Cell Engager (BiTE) antibody construct or chimeric antigen receptor T (CAR-T) cells. Blinatumomab, an FDA-approved BiTE, binds to CD19 on B cells and to CD3 on T cells, mediating effector-target cell contact and T-cell activation that results in effective elimination of target B cells. Although CD19 is expressed by essentially all B-cell malignancies at clinical presentation, relapses with loss or reduction in CD19 surface expression are increasingly recognized as a cause of treatment failure. Therefore, there is a clear need to develop therapeutics for alternate targets. We have developed a novel BiTE consisting of humanized anti-CD22 and anti-CD3 single chain variable fragments. Target binding of the anti-CD22 and anti-CD3 moieties was confirmed by flow cytometry. CD22-BiTE promoted in vitro cell-mediated cytotoxicity in a dose and effector: target (E:T)-dependent fashion. Additionally, in an established acute lymphoblastic leukemia (ALL) xenograft mouse model, CD22-BiTE demonstrated tumor growth inhibition, comparable to blinatumomab. Further, the combination of blinatumomab and CD22-BiTE yielded increased efficacy in vivo when compared to the single agents. In conclusion, we report here the development of a new BiTE with cytotoxic activity against CD22+ cells which could represent an alternate or complementary therapeutic option for B-cell malignancies.

A bispecific antibody AP203 targeting PD-L1 and CD137 exerts potent antitumor activity without toxicity

BackgroundBispecific antibody has garnered considerable attention in the recent years due to its impressive preliminary efficacy in hematological malignancies. For solid tumors, however, the main hindrance is the suppressive tumor microenvironment, which effectively impedes the activation of infiltrating T cells. Herein, we designed a bispecific antibody AP203 with high binding affinity to PD-L1 and CD137 and assessed its safety and anti-tumor efficacy, as well as explored the mechanism of action.MethodsThe optimal antibody binders against PD-L1 and CD137 were screened from the OmniMab phagemid library. The binding affinity of the constructed AP203 were evaluated using enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). T-cell stimulatory capacity was assessed using the allogeneic mixed lymphocyte reaction (MLR), antigen-specific recall response, and coculture with PD-L1-expressing cells. In vivo antitumor efficacy was evaluated using two models of tumor-xenografted humanized mice with profiling of tumor infiltrating lymphocytes (TILs). The possible toxicity of AP203 was examined using in vitro cytokine release assay by human PBMCs.ResultsAP203, which simultaneously targeted PD-L1 and costimulatory CD137, elicit superior agonistic effects over parental antibodies alone or in combination in terms of T cell activation, enhanced memory recall responses, and overcoming Treg-mediated immunosuppression (P < 0.05). The agonistic activity of AP203 was further demonstrated PD-L1-dependent by coculturing T cells with PD-L1-expressing cells. In vivo animal studies using immunodeficient or immunocompetent mice both showed a dose-related antitumor efficacy superior to parental antibodies in combination (P < 0.05). Correspondingly, AP203 significantly increased tumor infiltrating CD8 + T cells, while decreased CD4 + T cells, as well as Treg cells (P < 0.05), resulting in a dose-dependent increase in the CD8 + /CD4 + ratio. Moreover, either soluble or immobilized AP203 did not induce the production of inflammatory cytokines by human PBMCs.ConclusionsAP203 exerts potent antitumor activity not only by blocking PD-1/PD-L1 inhibitory signaling, but also by activating CD137 costimulatory signaling in effector T cells that consequently counteracts Treg-mediated immunosuppression. Based on promising preclinical results, AP203 should be a suitable candidate for clinical treatment of solid tumors.

Ångström-resolution fluorescence microscopy

Fluorescence microscopy, with its molecular specificity, is one of the major characterization methods used in the life sciences to understand complex biological systems. Super-resolution approaches1–6 can achieve resolution in cells in the range of 15 to 20 nm, but interactions between individual biomolecules occur at length scales below 10 nm and characterization of intramolecular structure requires Ångström resolution. State-of-the-art super-resolution implementations7–14 have demonstrated spatial resolutions down to 5 nm and localization precisions of 1 nm under certain in vitro conditions. However, such resolutions do not directly translate to experiments in cells, and Ångström resolution has not been demonstrated to date. Here we introdue a DNA-barcoding method, resolution enhancement by sequential imaging (RESI), that improves the resolution of fluorescence microscopy down to the Ångström scale using off-the-shelf fluorescence microscopy hardware and reagents. By sequentially imaging sparse target subsets at moderate spatial resolutions of >15 nm, we demonstrate that single-protein resolution can be achieved for biomolecules in whole intact cells. Furthermore, we experimentally resolve the DNA backbone distance of single bases in DNA origami with Ångström resolution. We use our method in a proof-of-principle demonstration to map the molecular arrangement of the immunotherapy target CD20 in situ in untreated and drug-treated cells, which opens possibilities for assessing the molecular mechanisms of targeted immunotherapy. These observations demonstrate that, by enabling intramolecular imaging under ambient conditions in whole intact cells, RESI closes the gap between super-resolution microscopy and structural biology studies and thus delivers information key to understanding complex biological systems.

Efficacy of Bispecific Antibody Targeting EpCAM and CD3 for Immunotherapy in Ovarian Cancer Ascites: An Experimental Study.

This study aimed to explore the value of M701, targeting epithelial cell adhesion molecule (EpCAM) and CD3, in the immunotherapy of ovarian cancer ascites by the in vitro assay. The expression of EpCAM in ovarian cancer tissues was analyzed by databases. The EpCAM expression and immune cell infiltration in different foci of ovarian cancer were detected by 8-channel flow cytometry. The toxic effect of M701 on OVCAR3 was tested using the in vitro cytotoxicity assay. The 3D cell culture and drug intervention experiments were performed to evaluate the therapeutic effect of M701 in ovarian cancer specimens. Flow cytometry was used to examine the effect of M701 on the binding of immune cells to tumor cells and the activation capacity of T cells. The results of the bioinformatic analysis showed that the expression of EpCAM in ovarian cancer tissue was significantly higher than that in normal ovarian tissue. The 8-channel flow cytometry of clinical samples showed that the EpCAM expression and lymphocyte infiltration were significantly heterogeneous among ovarian cancer patients and lesions at different sites. The in vitro experiment results showed that M701 had a significant killing effect on OVCAR3 cells. M701 also obviously killed primary tumor cells derived from some patients with ovarian cancer ascites. M701 could mediate the binding of CD3+ T cells to EpCAM+ tumor cells and induce T cell activation in a dose-dependent manner. M701 showed significant inhibitory activity on tumor cells derived from ovarian cancer ascites, which had a promising application in immunotherapy for patients with ovarian cancer ascites.

Pharmacodynamic-Mediated Drug Disposition (PDMDD) Model of Daratumumab Monotherapy in Patients with Multiple Myeloma.

We aimed to quantify the daratumumabconcentration- and CD38 dynamics-dependent pharmacokinetics using a pharmacodynamic mediated disposition model (PDMDD) in patients with multiple myeloma (MMY) following daratumumab IV or SC monotherapy. Daratumumab, a human IgG monoclonal antibody targeting CD38 with a direct on-tumor and immunomodulatory mechanism of action, has been approved to treat patients with multiple myeloma (MM). In total, 7788 daratumumab plasma samples from 850 patients with diagnosis of MMY were used. The serum concentration-time data of daratumumab were analysed using nonlinear mixed-effects modeling with NONMEM®. The PDMDD with quasi steady-state approximation (QSS) was compared to the previously developed Michaelis-Menten (MM) approximation with respect to the parameter estimates, the goodness-of-fit plots and prediction-corrected visual predictive check, as well as model-based simulations. The effect of patients' covariates on daratumumab pharmacokinetics was also investigated. The QSS approximation characterized the concentration- and CD38 dynamics-dependency of daratumumab pharmacokinetics within the doses ranging from 0.1 to 24 mg/kg after IV administration and 1200 and 1800 mg after SC administration in patients with MMY, mechanistically describing the binding of daratumumab and CD38, the internalization of the daratumumab-CD38 complex and the CD38 turnover. Compared to the previously developed MM approximation, the MM approximation with the non-constant total target and dose-correction provided substantial improvement of the model fit, but it was still not as good as the QSS approximation. The effect of the previously identified covariates as well as the newly identified covariate (baseline M protein) on daratumumab pharmacokinetics was confirmed, but the magnitude of the effect was deemed not clinically relevant. Accounting for the CD38 turnoverand its binding capacity to daratumumab, the QSS approximation provided a mechanistic interpretation of daratumumab PK parameters and consequently well described the concentration- and CD38 dynamics-dependency of daratumumab pharmacokinetics. CLINICAL STUDIES INCLUDED IN THE ANALYSIS WERE REGISTERED WITH THE NCT NUMBER BELOW AT HTTP://WWW. GOV : MMY1002 (ClinicalTrials.gov: NCT02116569), MMY1003 (NCT02852837), MMY1004 (NCT02519452), MMY1008 (NCT03242889), GEN501 (NCT00574288), MMY2002 (NCT01985126), MMY3012 (NCT03277105).

Therapeutic response to rituximab in seropositive neuromyelitis optica: Experience from a tertiary care center in South India.

Neuromyelitis optica (NMO) is a severe central nervous system demyelinating disease caused by autoantibodies to anti-aquaporin-4 immunoglobulin-G (AQP4-IgG). Rituximab, a monoclonal antibody targeting CD20 cells, is effective in neuromyelitis optica spectrum disorder (NMOSD) in several observational studies and small randomized controlled trials. However, this includes both AQP4-IgG antibody positive and negative cases. Whether rituximab is more effective in seropositive NMO is unknown. The aim of the study was to determine the efficacy of rituximab in seropositive NMO. This single-center ambispective study with retrospective data collection and prospective follow-up included patients with NMOSD who were positive for AQP4-Ig-G and treated with rituximab. Efficacy outcomes assessed were annualized relapse rate (ARR), disability progression by expanded disability status scale (EDSS), very good outcome (defined as no relapse and an EDSS ≤3.5), and persistent antibody positivity. Safety was also monitored. Between June 2017 and December 2019, 15 AQP4-IgG-positive cases were identified. The mean (± SD) age was 36 ± 17.9 years and 73.3% were females. Transverse myelitis followed by optic neuritis was the most common presentations. Rituximab was initiated after a median period of 19-weeks from the disease onset. The mean number of rituximab doses received was 6.4 ± 2.3. After a mean follow-up duration of 107 ± 74.7 weeks from the first dose of rituximab, ARR significantly reduced from 0.5 ± 0.9 to 0.02 ± 0.08, difference 0.48 ± 0.86 (95% confidence intervals [CI], 0.0009-0.96; P = 0.05). The number of relapses also reduced significantly from 0.6 ± 0.8-0.07 ± 0.26 , a difference of 0.53 ± 0.91 (95% CI, 0.026-1.05; P = 0.041). EDSS also significantly reduced from 5.6 ± 2.5-3.3 ± 2.9 , a difference of 2.23 ± 2.36 (95% CI, 0.93-3.54; P = 0.003). Very good outcome was obtained in 73.3% (11 of 15); P = 0.002. AQP4-IgG remained positive in 66.7% (4 of 6) when repeated after a mean period of 149.5 ± 51.1 weeks after the first dose of rituximab. Neither pre-treatment ARR, EDSS, time to initiate rituximab, the total number of rituximab doses, or time to repeat AQP4-IgG were significantly associated with persistent antibody positivity. No serious adverse events were observed. Rituximab exhibited high efficacy and good safety in seropositive NMO. Larger trials in this subgroup are warranted to confirm these findings.

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.

Abstract 2976: A single administration of DNA-encoded tri-specific T cell engager (DTriTE) targeting EGFRvIII, IL13Ra2, and CD3 shows complete tumor clearance in an orthotopic challenge model of heterogeneous GBM

Abstract Glioblastoma multiforme (GBM) is the most common and lethal form of primary brain tumors in adults with a five-year survival rate less than 5%. In recent years, immunotherapeutic approaches that target a tumor-associated antigen (TAA), including EGFRvIII-targeted cancer vaccine and adoptive cell transfer, have been examined in clinical studies, which so far have shown limited efficacy in GBM patients. GBM is highly heterogeneous for targeting antigen expression. An immunotherapeutic agent that harnesses multiple TAAs simultaneously may mitigate tumor antigen escape and improve efficacy in GBM patients. Here, we describe a DNA-encoded tri-specific T cell engager (DTriTE) that targets CD3 and two TAAs, EGFRvIII and IL13Ra2, which are expressed in up to 30% and 75% of GBM cases, respectively. We designed EGFRvIII-IL13Ra2-DTriTE to include single-chain variable fragments targeting IL13Ra2, CD3, and EGFRvIII in a series, fused with glycine-serine linkers. EGFRvIII-IL13Ra2-DTriTE exhibited robust binding activities to both EGFRvIII+ and IL13Ra2+ tumor cells and primary T cells, inducing robust T cell activation and anti-tumor cytotoxicity. EGFRvIII-IL13Ra2-DTriTE showed durable in vivo expression of over 10 weeks after a single IM injection in NSG mice. We developed an orthotopic challenge of a heterogeneous GBM model, wherein a mixture of EGFRvIII+ tumor cells and IL13Ra2+ tumor cells were planted in the brains of NSG mice and primary human T cells were peripherally delivered. A single IM administration of EGFRvIII-IL13Ra2-DTriTE resulted in complete tumor clearance. An administration of DNA-encoded bispecific T cell engager (DBTE) targeting EGFRvIII alone induced tumor clearance in 3 of 10 (30%) and limited tumor control in 5 of 10 (50%) animals. Similarly, mono-targeting of IL13Ra2 alone induced tumor clearance in 1 of 9 (11%) and limited tumor control in 4 of 9 (44%) animals. EGFRvIII-IL13Ra2-DTriTE treatment exhibited significantly improved survival with 8 of 8 (100%) animals survived, while treatments of single EGFRvIII-DBTE, IL13Ra2-DBTE or a vehicle control resulted in 4 of 10 (40%), 4 of 9 (44%), and 0 of 10 (0%) animals survived, respectively. This study shows that dual tumor-targeting approach using DTriTE is highly effective at treating antigenically heterogeneous GBM tumors. Further study of DTriTE multi-antigen approaches for GBM and other heterogeneous tumors appears important. Citation Format: Daniel Hongil Park, Pratik Bhojnagarwala, Kevin Liaw, Devivasha Bordoloi, Ebony N. Gary, David B. Weiner. A single administration of DNA-encoded tri-specific T cell engager (DTriTE) targeting EGFRvIII, IL13Ra2, and CD3 shows complete tumor clearance in an orthotopic challenge model of heterogeneous GBM [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 2976.

Abstract 3250: Targeting CD38 on exhausted T cells overcomes resistance to cancer immunotherapy

Abstract Despite the unprecedented success of immune checkpoint blockade (ICB) in melanoma and other cancers, therapeutic resistance remains a major challenge. CD38, an ecto-enzyme involved in NAD+ catabolism, is upregulated in dysfunctional/exhausted CD8+ T cells in human melanoma. As enrichment of dysfunctional CD8+ T cells is associated with lack of response to ICB, and NAD+ depletion via enhanced CD38 activity has been associated with diminished immune response, we hypothesized that CD38 might represent an attractive target to overcome resistance to ICB. While CD38 blockade has been shown to augment response to ICB in murine tumor models, the specific role of CD38 signaling in CD8 T cell exhaustion, and the therapeutic relevance of CD38 in human cancer have not been defined. Here, we confirm and extend previous observations that CD38 is enriched in exhausted CD8+ T cells during tumor progression and in response to ICB. Further, we demonstrate that disrupting CD38 signaling via neutralizing antibodies or pharmacological inhibition enhances response to ICB using established murine tumor models and patient-derived tumor explant models. Notably, we observed 54% response rate to combination PD-1/CD38 blockade using a cohort (n=35) of patient-derived organotypic tumor spheroids (PDOTS) from patients with ICB-refractory melanoma (11.4% response rate to single-agent PD-1 blockade). Supplementation of nicotinamide riboside (NR) to boost NAD+ levels mimicked the effects CD38 blockade/inhibition on PD-1 response, whereas FK866, an inhibitor of NAMPT a key step in NAD+ biosynthesis, blunted the response to combined PD-1/CD38 blockade, suggesting a major role for NAD+ in the efficacy of CD38 blockade/inhibition. Lastly, we demonstrate that in vitro CD38 inhibition/blockade in CD8+ T cells increases the levels of TCF7 (an effector/memory T cell transcription factor), decreases surface expression of co-inhibitory receptors (e.g., TIM-3, CD39, PD-1), and improves effector CD8 T cell function. Taken together, these data confirm a role for CD38 in CD8+ T cell exhaustion in melanoma and support further pre-clinical and clinical development of this novel therapeutic strategy to enhance anti-tumor immune responses. Citation Format: Or-Yam Revach, Angelina M. Cicerchia, Moshe Sade-Feldman, Seth Anderson, Robert T. Manguso, Nir Hacohen, Russell W. Jenkins. Targeting CD38 on exhausted T cells overcomes resistance to cancer immunotherapy [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 3250.

Abstract 1875: A novel T-cell-engaging therapeutical antibody against GPRC5D&amp;BCMA for multiple myeloma treatment

Abstract T-cell-engaging therapeutical antibodies, such as Talquetamab (targeting GPRC5D and CD3 with 1:1 formation) and CC-93269(targeting BCMA and CD3 with 2:1 formation) have shown promise in multiple myeloma (MM) treatment. However, MM usually had heterogeneous cell populations with independent expressions of BCMA and GPRC5D, so TCEs only targeting BCMA or GPRC5D are suspected to have limitation on anti-tumor effect. Development a TCE simultaneously binding BCMA and GPRC5D may extend its therapeutic efficacy. MBS314 is a humanized tri-specific antibody against GPRC5D, BCMA, and CD3 for r/r MM treatment. MBS314 has a weaker binding to CD3 (KD=181nM) than Talquetamab (KD=25.6 nM) and CC-93269 (KD=41.0 nM). Meanwhile,it has high affinities to GPRC5D (KD=3.5nM) and BCMA (KD=0.22nM), similar to Talquetamab (KD=4.21 nM, to GPRC5D) and CC-93269 (KD=1.0 nM, to BCMA). We developed a reporter assay to evaluate the function of TCEs: co-culture the tumor cells, such as H929, AMO1, or KMS-11, with Jurkat-NFAT-Luc reporter cells can induce luciferase expression, which can reflect the CD3 agonist signaling. MBS314 stimulated CD3 signaling when the reporter cells cultured along with any of the above MM cells, while Talquetamab failed to stimulate CD3 signaling when the reporter cells cultured along with KMS-11, which has low expression of GPRC5D, and CC-93269 was unable to stimulate CD3 signaling with AMO1 which has low expression of BCMA. The cytotoxicity assay also indicated MBS314 mediated T cell killing any of the above cells, while Talquetamab failed to kill KMS-11 and CC-93269 killed AMO-1 with low efficiency. In our in vivo xenograft MM murine models, MBS314 and Talquetamab induced regression of established MM.1S tumor in all of the mice (4/4), while CC-93269 induced regression in half of the mice (2/4) with dose at 0.5mg/kg injected twice a week. And MBS314 inhibited the growth of the MOLP8 tumor, while Talquetamab and CC-93269 failed to prevent MOLP8 tumor growth. Especially, our ex vivo cytotoxicity assay with bone marrow mononuclear cells (BM-MNCs) from 15 MM patients indicated that MBS314 more efficiently induced T-cell-mediated depletion of CD138+ MM cells than Talquetamab (mean(EC50)=0.06nM vs.1nM) and CC-93269 (mean(EC50)= 0.06nM vs.0.33nM). In addition, owning to the low affinity to CD3, MBS314 has no detectable toxicity in CD3 humanized mice. In summary, our data show MBS314 is a promising TCE for MM treatment with high efficacy and broad spectrum targeting both GPRC5D and BCMA, and also has excellent safety with low CD3 binding affinity. Citation Format: Feng Li, Guojin Wu, Hao Peng, Jiangmei Li, Yue Qi, Guangzhong Lin, Huifang Liu, Wenqi Hu, Jinying Ning. A novel T-cell-engaging therapeutical antibody against GPRC5D&amp;BCMA for multiple myeloma 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 1875.

Abstract 886: Fine-tuned CAIX targeted CAR-T cells exhibit superior efficacy and mitigate on-target off-tumor side effects

Abstract Chimeric Antigen Receptor (CAR) T cell therapy is a new type of “living drug” that has proven to be a powerful immunotherapy for hematologic malignancies. To date, there are six CAR-T products approved by the FDA for hematologic malignancies, four targeting CD19, and two targeting B-cell maturation antigen (BCMA). However, this success has not yet been transferred to solid tumors. A major hurdle is the on-target off-tumor toxicities due to the shared expression of target antigen on normal tissues. Carbonic anhydrase IX (CAIX) is highly expressed in clear cell renal cell carcinoma (ccRCC); however, it is also expressed on bile duct at a lower physiological level leading to off-tumor toxicity of CAIX targeted therapies. The first anti-CAIX CAR-T studies, using the 1st generation G250 CAR-T cells plus IL-2 to treat patients with metastatic ccRCC, caused severe liver enzyme abnormalities in the treated patients after CAR-T cell infusions. To understand CAIX expression on tumor and normal tissues, we quantified CAIX expression on ccRCC patient samples and healthy bile duct tissues using direct stochastic optical reconstruction microscopy (dSTORM) which provides single-molecule resolution. Tet-On inducible CAIX expressing cell lines were established to mimic various CAIX densities on normal tissue and tumor samples. Using biolayer interferometry (BLI) and avidity analyzer, we identified a low-affinity, high-avidity anti-CAIX CAR G9. G9 CAR-T cells only kill CAIX high ccRCC tumor cells but not CAIX low normal cholangiocytes, and exhibited a CAIX density dependent response to Tet-On inducible CAIX expressing cell lines. Compared to high-affinity G250 CAR-T cells, G9 showed a better safety profile and a wider therapeutic window. G9 demonstrated a superior ex vivo efficacy on ccRCC patient derived organotypic tumor spheroids (PDOTS) 3D cultures which recapitulate ccRCC patient tumor microenvironment (TME), as well as low toxicity on cholangiocyte derived organotypic spheroids (CDOS). In summary, affinity/avidity fine-tuned CAIX targeted CAR-T cell therapy holds promise to achieve cures of ccRCC by efficaciously killing tumor cells and mitigating on-target off-tumor toxicity on normal tissues. Citation Format: Yufei Wang, Alicia Buck, Gabriella Kastrunes, Rabia Abbas, Michael Lynch, Zhou Zhong, Song-My Hoang, Andras Miklosi, Kun Huang, Jae-Won Cho, Marion Grimaud, Cecile Razimbaud, Matthew Chang, Atef Fayed, Audrey Apollon, Nithyassree Murugan, Ze-Hua Li, Tran Thai, Luann Zerefa, Brandon Piel, Elena Ivanova, Amy Cameron, Quang-De Nguyen, Zhu Zhu, Kevin Wei, Yasmin Nabil Laimon, Aseman Bagheri Sheshdeh, Sabina Signoretti, David A. Braun, Catherine J. Wu, Toni K. Choueiri, Jon Wee, Cloud P. Paweletz, Martin Hemberg, Aedin C. Culhane, David A. Barbie, Gordon J. Freeman, Wayne A. Marasco. Fine-tuned CAIX targeted CAR-T cells exhibit superior efficacy and mitigate on-target off-tumor side effects [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 886.