Dependent Cell-mediated Cytotoxicity Research Articles

Sequence Matters: Primary COVID-19 Vaccination after Infection Elicits Similar Anti-spike Antibody Levels, but Stronger Antibody Dependent Cell-mediated Cytotoxicity than Breakthrough Infection.

Infection before primary vaccination (herein termed "hybrid immunity") engenders robust humoral immunity and broad Ab-dependent cell-mediated cytotoxicity (ADCC) across SARS-CoV-2 variants. We measured and compared plasma IgG and IgA against Wuhan-Hu-1 and Omicron (B.1.1.529) full-length spike (FLS) and receptor binding domain after three mRNA vaccines encoding Wuhan-Hu-1 spike (S) and after Omicron breakthrough infection. We also measured IgG binding to Wuhan-Hu-1 and Omicron S1, Wuhan-Hu-1 S2 and Wuhan-Hu-1 and Omicron cell-based S. We compared ADCC using human embryonic lung fibroblast (MRC-5) cells expressing Wuhan-Hu-1 or Omicron S. The effect of Omicron breakthrough infection on IgG anti-Wuhan-Hu-1 and Omicron FLS avidity was also considered. Despite Omicron breakthrough infection increasing IgG and IgA against FLS and receptor binding domain to levels similar to those seen with hybrid immunity, there was no boost to ADCC. Preferential recognition of Wuhan-Hu-1 persisted following Omicron breakthrough infection, which increased IgG avidity against Wuhan-Hu-1 FLS. Despite similar total anti-FLS IgG levels following breakthrough infection, 4-fold higher plasma concentrations were required to elicit ADCC comparable to that elicited by hybrid immunity. The greater capacity for hybrid immunity to elicit ADCC was associated with a differential IgG reactivity pattern against S1, S2, and linear determinants throughout FLS. Immunity against SARS-CoV-2 following Omicron breakthrough infection manifests significantly less ADCC capacity than hybrid immunity. Thus, the sequence of antigenic exposure by infection versus vaccination and other factors such as severity of infection affect antiviral functions of humoral immunity in the absence of overt quantitative differences in the humoral response.

Abstract 117: Improved PBMC humanized mouse models for immuno-oncology drugs evaluation

Abstract It has been demonstrated that anti-PD(L)1 therapy could significantly improve survival outcomes for patients with metastatic solid cancers, such as melanoma, non-small cell lung cancer and MSI-H colorectal cancer. However, more than half of these patients (NCT02563002, NCT02142738, NCT02563002) do not respond to immune checkpoint blockade (ICB) therapy, and 90% of colorectal cancer patients have MSS/pMMR characteristics and almost no response at all, which drives scientists to design novel drug candidates urgently and integrate tumor and tumor microenvironment (TME) signals systemically to rejuvenate exhausted immune system and overcome ICB resistance. LIDE has developed both traditional PBMC humanized mice model, and human immune cells and cancer co-inoculated model for IO drugs functional evaluation in the last 5-years by using unique PBMC donor selection and standard cancer biobank (PDX patient-derived xenografts, CDX cell derived xenograft) establishment. Cancer biopsies with MDM2 amplification or DNMT3A alternation have been shown resistant to PD1-Ab therapy but sensitive to chemical combo-therapy, and pMMR cancer PDX models seem responsible well to EGFR targeted, CD39-CD73-Adenosine targeted therapy. Human IL15 transgenic mice are used as one useful tool to investigate in vivo function of NK cells, NK engagers/modulators as well as drugs with ADCC (Antibody dependent cell-mediated cytotoxicity) effect. The activated immune cells and cancer co-transfer model, can significantly delay the graft-versus-host-reaction for another month compared with traditional PBMC humanized model, and archives maximal therapeutic effect of the testing drugs by pre-activating T cells with specific tumor antigens. Dendritic cells or macrophages could be successfully introduced into this co-inoculated system to work together with T cells, mimic human tumor tissue complexity, to help determine the overall effect induced by integrated signals. It seems CD40 agonist or CD47 antagonist could well synergies with anti-PD(L)1 to control melanoma and triple negative breast cancer development, respectively. This optimized PBMC humanized cancer-bearing mice models have been tested for various cancer targets, e.g. PD(L)1, Tim3, Lag3, Tigit, PVRIG, TGFβ1, CD73/CD39, CD47/Sirpa, DLL3/CD3, Claudin18.2/4-1BB, B7-H3, GITR, to better understand the interaction between cancer cells and immune cells. Citation Format: Bin Xie, Xin Hou, Pengfei Yang, Yanbing Zhou, Danyi Wen. Improved PBMC humanized mouse models for immuno-oncology drugs evaluation [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 117.

Development and implementation of natural killer cell simultaneous ADCC and direct killing assay

Assays to quantify natural killer (NK) cell killing efficacy have traditionally focused on assessing either direct killing or antibody dependent cell-mediated cytotoxicity (ADCC) independently. Due to the probability that immunotherapeutic interventions affect NK cell-mediated direct killing and NK cell-mediated ADCC differently, we developed an assay with the capacity to measure NK cell-mediated direct killing and ADCC simultaneously with cells from the same human donor. Specifically, this design allows for a single NK cell population to be split into several experimental conditions (e.g., direct killing, ADCC), thus controlling for potential confounders associated with human-to-human variation when assessing immunotherapy impacts. Our Natural Killer cell Simultaneous ADCC and Direct Killing Assay (NK-SADKA) allows researchers to reproducibly quantify both direct killing and ADCC by human NK cells. Furthermore, this optimized experimental design allows for concurrent analysis of the NK cells via flow cytometric immunophenotyping of NK cell populations which will facilitate the identification of relationships between NK cell phenotype and the subsequent killing potential. This assay will be valuable for assessing the broader impact(s) of immunotherapy strategies on both modes of NK cell killing.

Antibody dependent cell-mediated cytotoxicity selection pressure induces diverse mechanisms of resistance

ABSTRACT Targeted monoclonal antibody therapy has emerged as a powerful therapeutic strategy for cancer. However, only a minority of patients have durable responses and the development of resistance remains a major clinical obstacle. Antibody-dependent cell-mediated cytotoxicity (ADCC) represents a crucial therapeutic mechanism of action; however, few studies have explored ADCC resistance. Using multiple in vitro models of ADCC selection pressure, we have uncovered both shared and distinct resistance mechanisms. Persistent ADCC selection pressure yielded ADCC-resistant cells that are characterized by a loss of NK cell conjugation and this shared resistance phenotype is associated with cell-line dependent modulation of cell surface proteins that contribute to immune synapse formation and NK cell function. We employed single-cell RNA sequencing and proteomic screens to interrogate molecular mechanisms of resistance. We demonstrate that ADCC resistance involves upregulation of interferon/STAT1 and DNA damage response signaling as well as activation of the immunoproteasome. Here, we identify pathways that modulate ADCC sensitivity and report strategies to enhance ADCC-mediated elimination of cancer cells. ADCC resistance could not be reversed with combinatorial treatment approaches. Hence, our findings indicate that tumor cells utilize multiple strategies to inhibit NK cell mediated-ADCC. Future research and development of NK cell-based immunotherapies must incorporate plans to address or potentially prevent the induction of resistance.

Structure and Fc-Effector Function of Rhesusized Variants of Human Anti-HIV-1 IgG1s.

Passive transfer of monoclonal antibodies (mAbs) of human origin into Non-Human Primates (NHPs), especially those which function predominantly by a Fc-effector mechanism, requires an a priori preparation step, in which the human mAb is reengineered to an equivalent NHP IgG subclass. This can be achieved by changing both the Fc and Fab sequence while simultaneously maintaining the epitope specificity of the parent antibody. This Ab reengineering process, referred to as rhesusization, can be challenging because the simple grafting of the complementarity determining regions (CDRs) into an NHP IgG subclass may impact the functionality of the mAb. Here we describe the successful rhesusization of a set of human mAbs targeting HIV-1 envelope (Env) epitopes involved in potent Fc-effector function against the virus. This set includes a mAb targeting a linear gp120 V1V2 epitope isolated from a RV144 vaccinee, a gp120 conformational epitope within the Cluster A region isolated from a RV305 vaccinated individual, and a linear gp41 epitope within the immunodominant Cys-loop region commonly targeted by most HIV-1 infected individuals. Structural analyses confirm that the rhesusized variants bind their respective Env antigens with almost identical specificity preserving epitope footprints and most antigen-Fab atomic contacts with constant regions folded as in control RM IgG1s. In addition, functional analyses confirm preservation of the Fc effector function of the rhesusized mAbs including the ability to mediate Antibody Dependent Cell-mediated Cytotoxicity (ADCC) and antibody dependent cellular phagocytosis by monocytes (ADCP) and neutrophils (ADNP) with potencies comparable to native macaque antibodies of similar specificity. While the antibodies chosen here are relevant for the examination of the correlates of protection in HIV-1 vaccine trials, the methods used are generally applicable to antibodies for other purposes.

2O Penpulimab, an IgG1 anti-PD-1 antibody with Fc-engineering to eliminate effector functions and with unique epitope and binding properties

Currently marketed anti-PD-1 antibodies are typically IgG4 isotype, of which bindings to FcγRs and CH3 region instability might lead to compromise on efficacy and safety. Penpulimab is a humanized IgG1 anti-PD-1 antibody engineered at the Fc region to eliminate binding to FcγRs and C1q, and thus avoid antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) to minimize lymphocyte loss, and to avoid antibody dependent cytokine release (ADCR) such as IL-8 and IL-6, which are known to associate with irAEs and poor prognosis in checkpoint blocking immunotherapy.

3D tumor spheroid microarray for high-throughput, high-content natural killer cell-mediated cytotoxicity

Immunotherapy has emerged as a promising approach to treating several forms of cancer. Use of immune cells, such as natural killer (NK) cells, along with small molecule drugs and antibodies through antibody dependent cell-mediated cytotoxicity (ADCC) has been investigated as a potential combination therapy for some difficult to treat solid tumors. Nevertheless, there remains a need to develop tools that support co-culture of target cancer cells and effector immune cells in a contextually relevant three-dimensional (3D) environment to provide a rapid means to screen for and optimize ADCC-drug combinations. To that end, here we have developed a high throughput 330 micropillar-microwell sandwich platform that enables 3D co-culture of NK92-CD16 cells with pancreatic (MiaPaCa-2) and breast cancer cell lines (MCF-7 and MDA-MB-231). The platform successfully mimicked hypoxic conditions found in a tumor microenvironment and was used to demonstrate NK-cell mediated cell cytotoxicity in combination with two monoclonal antibodies; Trastuzumab and Atezolizumab. The platform was also used to show dose response behavior of target cancer cells with reduced EC50 values for paclitaxel (an anti-cancer chemotherapeutic) when treated with both NK cells and antibody. Such a platform may be used to develop more personalized cancer therapies using patient-derived cancer cells.

Dinitrophenol-mediated modulation of an anti-PD-L1 VHH for Fc-dependent effector functions and prolonged serum half-life

Single-domain antibodies, VHHs or nanobodies, represent a promising set of alternatives to conventional therapeutic antibodies, gaining substantial attention in the field of cancer immunotherapy. However, inherent drawbacks of nanobodies such as fast clearance from blood circulation and lack of immune effector functions often led to unsatisfactory therapeutic efficacy. We previously reported that dinitrophenyl modification of an anti-EGFR VHH conferred Fc-dependent immune effector functions and elongated serum half-life on it through recruiting of hapten antibodies, resulting in improved immunotherapy efficacy in vivo. In the present work, we further tested the versatility of this approach in the case of an anti-PD-L1 blockade VHH (KN035). Site-specific dinitrophenyl conjugation did not impair the binding capacity of KN035 portion to PD-L1, but indirectly restored its immune effector functions, manifested by the observed antibody dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and complement-dependent cytotoxicity against PD-L1 positive tumor cells. Significant delay of blood clearance of dinitrophenylated KN035 was evidenced by the prolonged half-life of ca. 22 h. This approach, using small hapten molecule conjugation, loaded additional antibody-mediated tumor killing mechanisms to PD-L1 blockade VHH and therefore improved efficacy is anticipated in the future in vivo therapeutic studies. Thus, our results underscore the power of this versatile approach for achieving desirable properties of VHH-based or similar therapeutics.

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

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

Abstract 1843: Dicovery and characterization of a novel anti-human CXCR5 antibody for the treatment of B cell lymphomas

Abstract CXCR5 is a seven transmembrane protein coupled to protein G(i) expressed mainly on mature, resting B cells, follicular helper T cells (TFH), tonsillar B cells, and B cell lymphoma cells. CXCR5 is activated by its ligand CXCL13 and involved in cell migration. Dysregulated CXCL13 and/or CXCR5 expression and signaling have been associated with cancer promotion, as well as autoimmune and chronic inflammatory disorders. Overexpression of CXCL13 in autoimmune and lymphoproliferative disorders has been shown to correlate with disease progression. We therefore hypothesized that an antibody that selectively depletes CXCR5+ malignant B cells through antibody dependent cell-mediated cytotoxicity (ADCC) could be developed to treat B cell lymphomas. Here, we present preclinical data supporting the rationale for indication selection, biomarker identification, dose selection, and phase I clinical trial design. Using our single-cell screening platform technology, CelliGo™, we identified a panel of monoclonal antibodies against human CXCR5. We selected HFB2-4, which binds to CXCR5 expressing cells, blocks CXCL13-induced intracellular signaling and B cell migration, and induces ADCC of Raji B cell lymphoma cells at sub-nM concentrations. HFB2-4 demonstrates good PK behavior in mice and exhibits potent anti-tumor activity in a Raji xenograft murine model. HFB2-4 efficacy in this model is comparable to that of rituximab, despite a lower number of huCXCR5 receptors compared to CD20 on the surface of Raji cells. Our data shows that HFB2-4 holds promise as a therapeutic agent for B cell lymphoproliferative diseases by depleting CXCR5+ tumor B cells in refractory B cell lymphoma and lymphomas derived from severe Sjogren syndrome. Compared to the emerging treatments for refractory B cell lymphomas such as Antibody-Drug-Conjugates, bi-specific antibodies or cell therapies, HFB2-4 may represent a well-tolerated, efficacious, cost-efficient and easy to manufacture alternative. Citation Format: Ayrin Kok, Germain Margallducos, Stephanie Beq, Clarisse Monchecourt, Hombline Poullain, Muriel David, Carine George, Sami Ellouze, Mingjie Chen, Yun-Yueh Lu, Juying Li, Qian Zhang, Jean Wang, Francisco Adrian, Liang Schweizer, Nicola Beltraminelli. Dicovery and characterization of a novel anti-human CXCR5 antibody for the treatment of B cell lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1843.

Lenalidomide and Obinutuzumab with CHOP for Newly Diagnosed Diffuse Large B-Cell Lymphoma: Final Phase I/II Results

▪Introduction:Diffuse large B-cell Lymphoma (DLBCL) is the most common lymphoid cancer in the United States with 30K diagnoses each year. The standard therapy for all newly diagnosed patients is rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP) which cures ~60% of patients. Lenalidomide (L) and obinutuzumab (O) have both shown promising efficacy and acceptable toxicity in DLBCL patients. Obinutuzumab, a humanized CD20 monoclonal antibody with increased pre-clinical affinity to the FcGRIIIa receptor in comparison to rituximab, may have increased antibody dependent cell-mediated cytotoxicity (ADCC). Lenalidomide, a potent immunomodulatory, is toxic to DLBCL via interferon signaling stimulation. As single agents, O and L achieve overall response rates (ORR) of 32% and 28%, respectively, in patients with relapsed DLBCL (Morschhauser, 2013, Hernandez-Ilizaliturri, 2011). The addition of L to RCHOP may overcome adverse outcomes of the non-germinal center (non-GCB) subtype of DLBCL. Randomized phase III clinical trials evaluating RCHOP +/- Len in non-GCB DLBCL are highly anticipated. The GOYA study found RCHOP was as effective as OCHOP in patients with unselected newly diagnosed DLBCL. We hypothesize the combination of OL-CHOP will result in enhanced NK cell-mediated cytotoxicity, down-regulation of interferon regulatory factor 4, and a complete response (CR) rate ≥ to OCHOP (57% in GOYA trial, Vitolo et al, ASH 2016).Methods: Newly diagnosed, CD20+ DLBCL patients were eligible if they have measurable disease, age ≥18 years, and adequate organ function. In the Phase Ib trial, Obinutuzumab was dosed at 1000mg IV days (d) 1, 8, and 15 during cycle 1, and d 1 on cycles 2-6. Lenalidomide dose was 15mg d 1 - 14 cycles 1 - 6 in the first cohort, with lower dose cohorts if toxicities occurred, using a 3+3 design. CHOP was dosed in standard fashion. Once the maximum tolerated dose (MTD) was identified, phase II was initiated with a planned total of at least 50 evaluable patients at the MTD. The primary objectives of this phase Ib/II trial were to determine the MTD and efficacy of LOCHOP. Correlative studies, including minimal disease assessment, cell of origin determination, and immune profiling are planned on blood and tumor samples.Results: Fifty three patients were enrolled, 6 in Phase Ib trial, 47 in Phase II with all evaluable for safety and 51 for efficacy. Two patients withdrew consent after 1 cycle of therapy. The median age was 62 years (26-83), with 42% males and a median IPI of 2 and NCCN IPI of 3. Five patients had double hit DLBCL, 5 patients had Double Expressor DLBCL, and 21 patients were non-GCB DLBCL. No dose limiting toxicities were identified in in Phase Ib. Toxicities encountered in PhIb include neutropenia (Grade 3: 50%, Grade 4 33%), thrombocytopenia (grade 3: 17%), and rash (grade 2: 17%), with similar findings in PhII. All six patients in PhIb achieved a complete response (CR). In the Phase II trial, 50 patients have end of therapy response assessment as of 8/2/17, with 45 with CR, 4 with partial response, 1 with progression. In the combined evaluable patients, the overall response rate is 98%, with CR of 90%. The CR rate was 90% in non-GCB and 92% in GCB. The median follow up 11 months and the median progression free and overall survival are not reached. There have been no deaths. Additional follow up and analyses will be presented at the meeting.Conclusions: The combination of Obinutuzumab, Lenalidomide, and CHOP is well tolerated, with no dose limiting toxicity encountered in the phase Ib trial, and efficacy is impressive in this single center single arm Phase Ib/II trial. Adverse events do not appear to be different than expected with standard RCHOP. Final results and correlative analyses will be presented at the ASH meeting [Display omitted] DisclosuresWestin:Apotex: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Nastoupil:TG Therapeutics: Honoraria, Research Funding; Gilead: Honoraria; Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Karus Therapeutics: Research Funding; Celgene: Honoraria, Research Funding. Neelapu:Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Poseida Therapeutics, Inc: Research Funding; Cellectis Inc.: Research Funding; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Karus: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Lee:KITE PHARMA: Consultancy.

Structural basis for RIFIN-mediated activation of LILRB1 in malaria.

The Plasmodium species that cause malaria are obligate intracellular parasites, and disease symptoms occur as they replicate within human blood. Despite risking immune detection, the parasite delivers proteins that bind host receptors to infected erythrocyte surfaces. In the causative agent of the most deadly human malaria, Plasmodium falciparum, RIFINs form the largest erythrocyte surface protein family1. Some RIFINs can bind inhibitory immune receptors, acting as targets for unusual antibodies containing a LAIR1 ectodomain2-4, or as ligands for LILRB15. RIFINs stimulate LILRB1 activation and signalling5, thereby potentially dampening human immune responses. To understand this process, we determined a structure of a RIFIN bound to LILRB1. We show that the RIFIN mimics the natural activating ligand of LILRB1, MHC class I, in its LILRB1-binding mode. A single RIFIN mutation disrupts the complex, blocks LILRB1 binding by all tested RIFINs and abolishes signalling in a reporter assay. In a supported lipid bilayer system, which mimics NK cell activation by antibody- dependent cell-mediated cytotoxicity, both RIFIN and MHC are recruited to the NK cell immunological synapse and reduce cell activation, as measured by perforin mobilisation. Therefore, LILRB1-binding RIFINs mimic the binding mode of the natural ligand of LILRB1 and suppress NK cell function.

Fully automated integrated process for depletion of T cells with or without subsequent purification of NK cells from apheresis using the CliniMACS Prodigy

Background & Aim Donor-derived natural killer (NK) cells mediate graft-versus-leukemia effects low risk in inducing alloreactive graft-versus-host-disease and are used as component of T cell-depleted donor lymphocyte infusion (DLI) or purified. As clinical grade purification of NK cells requires two semi-automated separation procedures, including multiple manual hands-on steps, we developed an integrated fully automated process enabling either preparation of T cell-depleted DLI or clinical grade purification of NK cells within a single automated process on the CliniMACS Prodigy. Methods, Results & Conclusion The novel integrated CliniMACS Prodigy LP-3-56 System uses the CliniMACS Prodigy Tubing Set 320 for an fully automated procedure with integrated labeling and washing steps for both, CD3 and CD56 separation enabled through an integrated lytic rinsing sequence allowing subsequent CD56 enrichment in the same tubing set. By users choice the depletion of CD3+ cells will be performed with or without subsequent enrichment of CD56+ cells. For both strategies target cells are prepared with user-chosen formulation solution (e. g. sodium chloride or NK MACS medium). The new application software effectively depletes 9.6 × 109 CD3+ cells, and enriches 4.5 × 109 CD56+ cells from up to 40 × 109 total white blood cells. Inhouse evaluation runs with leukapheresis products (n= 29 in total, n=17 with CD3 depletion, n=12 with CD3 depletion and CD56 enrichment) were performed. After CD3 depletion, mean log depletion of T cells was 4.0. The mean recovery of NK cells was 89% with an average NK:T ratio-increase of 9509 fold. After CD3 depletion and CD56 enrichment, mean log depletion of T and B cells was 4.2 and 2.7, respectively. Purity of NK cells was 90.7% on average with a mean recovery of 49%. The majority of non-NK cells after the process were monocytes with a mean frequency of 5.9%. Functionality of purified NK cells was proven by their ability to lyse AML cells, to secrete cytokines after co-culture, and to perform antibody dependent cell-mediated cytotoxicity (ADCC) against Raji cells, using human CD20 antibody. In summary, the integrated fully automated CliniMACS Prodigy LP-3-56 System is capable of efficiently deplete CD3+ cells with or without subsequent CD56 enrichment to purify functional NK cells from leukapheresis products and provides target cells in formulation solution. The performance verification and submission to an European notified body for CE mark are the important next steps.

Engineering of Fc Multimers as a Protein Therapy for Autoimmune Disease.

The success of Intravenous Immunoglobulin in treating autoimmune and inflammatory processes such as immune thrombocytopenia purpura and Kawasaki disease has led to renewed interest in developing recombinant molecules capable of recapitulating these therapeutic effects. The anti-inflammatory properties of IVIG are, in part, due to the Fc region of the IgG molecule, which interacts with activating or inhibitory Fcγ receptors (FcγRs), the neonatal Fc Receptor, non-canonical FcRs expressed by immune cells and complement proteins. In most cases, Fc interactions with these cognate receptors are dependent upon avidity—avidity which naturally occurs when polyclonal antibodies recognize unique antigens on a given target. The functional consequences of these avid interactions include antibody dependent cell-mediated cytotoxicity, antibody dependent cell phagocytosis, degranulation, direct killing, and/or complement activation—all of which are associated with long-term immunomodulatory effects. Many of these immunologic effects can be recapitulated using recombinant or non-recombinant approaches to induce Fc multimerization, affording the potential to develop a new class of therapeutics. In this review, we discuss the history of tolerance induction by immune complexes that has led to the therapeutic development of artificial Fc bearing immune aggregates and recombinant Fc multimers. The contribution of structure, aggregation and N-glycosylation to human IgG: FcγR interactions and the functional effect(s) of these interactions are reviewed. Understanding the mechanisms by which Fc multimers induce tolerance and attempts to engineer Fc multimers to target specific FcγRs and/or specific effector functions in autoimmune disorders is explored in detail.

Abstract C028: Discovery and characterization of next generation monoclonal antibodies targeting the inhibitory Fc gamma receptor CD32b for the treatment of B and plasma cell malignancies

Abstract CD32b (FcγR2b), the sole inhibitory Fcγ receptor, negatively regulates immune function and is expressed throughout B cell development and on their malignant counterparts with the highest expression found on multiple myeloma. Additionally, CD32b expression on tumor cells is known to sequester IgG Fc thereby providing a mechanism of resistance to therapeutic monoclonal antibodies (mAb) with Fc dependent activity. Taken together, CD32b represents an attractive tumor antigen for targeting with a mAb. To this end, two anti-CD32b mAbs, NVS32b1 and NVS32b2, were developed. The complementarity-determining regions (CDRs) of these antibodies bind the CD32b Fc binding domain with high specificity and affinity while the Fc region is afucosylated enabling enhanced activation of FcγR on immune effector cells. This specificity and optimized potency is highlighted in whole blood assays where NVS32b2 depletes CD32b positive B cells but spares immune subsets with low CD32b expression or expression of the homologous CD32a. The antibodies mediate potent killing of opsonized cells via antibody dependent cell-mediated cytotoxicity, antibody dependent cellular phagocytosis, and complement dependent cytotoxicity. Additionally, NVS32b mAbs’ CDR block the CD32b Fc binding domain, thereby minimizing CD32b mediated resistance to therapeutic mAbs with Fc dependent activity including rituximab, obinutuzumab, and daratumumab. In vivo, NVS32b mAbs demonstrate robust antitumor activity against CD32b positive xenografts and immunomodulatory activity including recruitment of intratumoral macrophages. The NVS32b mAbs’ activity against malignant B and plasma cells featuring a range of CD32b expression demonstrates their therapeutic potential, as a single agent or in combination with therapeutics including mAbs with Fc dependent activity. Citation Format: Haihui Lu, Dongshu Chen, Sunyoung Jang, Babette Wolf, Stefan Ewert, Meghan Flaherty, Fangmin Xu, Sinan Isim, Yeonjiu Shim, Christina Dornelas, Nicole Balke, Xavier Charles Leber, Meike Scharenberg, Johanna Koelln, Eugene Choi, Rebecca Ward, Jennifer Johnson, Thomas Calzascia, Isabelle Isnardi, Juliet Williams, Heather Huet, Emma Lees, Matthew J Meyer. Discovery and characterization of next generation monoclonal antibodies targeting the inhibitory Fc gamma receptor CD32b for the treatment of B and plasma cell malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C028. doi:10.1158/1535-7163.TARG-19-C028

Abstract 2970: Comparison of anti-CD38 antibodies in vitro mechanisms of action in multiple myeloma

Abstract Background: The outcome of multiple myeloma (MM) patients has been dramatically improved by the monoclonal antibody (mAb) daratumumab (DARA). DARA is a human IgG1κ mAb specific for CD38 and approved for the treatment of newly diagnosed and relapsed/refractory MM as a monotherapy and in combination with standard of care. Isatuximab and TAK-079 are additional CD38 targeting mAbs in clinical development by Sanofi and Takeda, respectively. These mAbs are reported to bind different epitopes and induce lysis of MM tumor cells via multiple mechanisms. It is unclear how the pleiotropic mechanisms collectively impact tumor cytolysis and exhibit anti-tumor effects in a comprehensive ex vivo immune milieu. Methods: For research purposes, DARA was compared with representative mAbs based on the published patent sequences for isatuximab and TAK-079, these surrogate analogs will hereafter be referred to as Sanofi anti-CD38 and Takeda anti-CD38. Mechanism of action (MOA) studies were done to compare antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cell phagocytosis (ADCP), complement mediated cytotoxicity (CDC), and early and late detection of apoptosis. In addition, fresh whole blood from healthy donors was used to assess the cumulative effect of the MOAs on MM cell lines. Results: We tested the CDC activity of the anti-CD38s on multiple MM cell lines with a range of CD38 surface expression and CDC sensitivity levels. In LP-1 and MOLP-8 MM cell lines, DARA resulted in higher levels CDC activity as compared to the other anti-CD38 mAbs. In ADCC and ADCP assays, all 3 anti-CD38 mAbs induced similar levels of MM cell death and phagocytosis. Apoptosis was also assessed in the presence and absence of FcR crosslinking. The Annexin V assay replicated published results that only the Sanofi anti-CD38 was able to induce phosphatidylserine translocation to the cell surface without FcR crosslinking. However, in a robust 5-day cytotoxicity assay detecting metabolically active cells, all 3 antibodies elicited comparable high levels of cell death in the presence of the FcR crosslinker and low levels in its absence. We also performed assays with fresh whole blood from healthy volunteers (n=6) and determined the cumulative effect of the anti-CD38 mAbs on LP-1 and MOLP-8 MM cell lines. DARA demonstrated a higher maximal cytotoxicity than the other mAbs. Moreover, DARA had a significantly lower EC50 than Takeda anti-CD38 in both cell lines and lower than Sanofi-anti-CD38 in MOLP-8. Conclusion: DARA and surrogate analogs of Sanofi anti-CD38 and Takeda anti-CD38 have similar MOAs with the exception of higher CDC demonstrated by DARA which may contribute to the observed improved efficacy of DARA in a comprehensive immune milieu in vitro. As DARA is the only approved CD38 mAb, it remains to be determined in clinical trials if these in vitro differences lead to differences in clinical benefit. Citation Format: Michelle Kinder, Mi Ta, Amy Axel, Amy Wong, Stephen Rudnick, Bart de Goeij, Jeroen Lammerts van Bueren, Alex Babich, Mark Mendonca, Jocelyn Sendecki, Christopher Chiu, Kevin Bellew, Colleen Kane. Comparison of anti-CD38 antibodies in vitro mechanisms of action in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2970.

Synergistic activity between IL-15 signaling and checkpoint blockade drives enhanced NK cell expansion and tumor control in ovarian cancer

Abstract Survival in ovarian cancer patients has not improved in the past 40 years, stressing a need for new lines of therapy. NK cell immunotherapy in ovarian cancer has not been extensively explored due to limited data showing defective NK cell function in these patients. Our previous work shows that an IL-15 super-agonist (NANT N-803 (formerly ALT-803)) can bypass some of the deficiencies. However, we show here that though N-803 enhances NK cell activation, it also induces NK PD-1 expression and inflammatory cytokines secreted by the NK cells induce PD-L1 expression on ovarian tumors. To bypass this negative feedback we combined N-803 with an anti-PD-L1 antibody (Avelumab) and show a potent increase in NK cell activation and long-term real-time in vitro tumor killing. Furthermore, combined N-803/Avelumab induces robust killing of ovarian cancer tumor spheroids, not seen with individual treatments. To evaluate the benefits of this combined treatment in vivo we tested them in a xenogeneic model of ovarian cancer containing human ovarian cancer cells (MA148lucs or OVCAR8lucs) and human NK cells. Our data clearly shows reduced tumor burden, by bioluminescent imaging, and potently increased NK cell expansion with the combined treatment when compared to individual treatments. Since Avelumab is capable of driving antibody dependent cell-mediated cytotoxicity (ADCC) we wanted to evaluate the impact of checkpoint signals in the synergy excluding ADCC using an anti-PD-1 antibody (Pembrolizumab). Combination of N-803 with Pembrolizumab yielded similar tumor control and NK cell expansion in the in vivo model, indicating that the synergy lies in checkpoint signals and IL-15 signaling, and not just in amplification of ADCC via N-803.

56P - HER2-targeted antibody dependent cell-mediated cytotoxicity (ADCC) mediated by NK cells against tyrosine kinase inhibitor (TKI)-treated breast cancer (BC) cell lines

56P - HER2-targeted antibody dependent cell-mediated cytotoxicity (ADCC) mediated by NK cells against tyrosine kinase inhibitor (TKI)-treated breast cancer (BC) cell lines

Abstract P6-18-11: Long-term responders to single-agent margetuximab, an Fc-modified anti-HER2 monoclonal antibody, in metastatic HER2+ breast cancer patients with prior anti-HER2 therapy

Abstract Background Margetuximab is an Fc-optimized anti-HER2 antibody that recognizes the same epitope as trastuzumab. Margetuximab has increased affinity for the activating CD16A Fc-receptor on NK cells and macrophages as well as decreased affinity for the inhibitory CD32B receptor compared to trastuzumab. In a Phase 1 study (NCT01148849) of 66 patients with relapsed or metastatic HER2+ cancer across multiple indications, margetuximab was well tolerated at all doses. Among 60 response-evaluable patients, confirmed partial response (PR) and stable disease (SD) were seen in 7 (12%) and 30 (50%) patients, respectively. Tumor reductions occurred in 18/23 (78%) evaluable breast cancer patients. Ex-vivo analyses of patient peripheral blood mononuclear cell samples confirmed margetuximab's ability to enhance antibody dependent cell-mediated cytotoxicity over that from trastuzumab. We report on 3 breast cancer patients with prior anti-HER2 therapy failure with durable (≥ 3.5 years) SD (1) or PR (2). Methods Enrolled patients had histologically/cytologically-confirmed carcinoma with documented HER2 overexpression by immunohistochemistry (2+ or 3+) and disease progression during/following last therapy. Eligibility included life expectancy ≥3 months; performance status ≤1; measurable disease by Response Criteria for Solid Tumors 1.1; adequate bone marrow, renal, hepatic function; and left ventricular ejection fraction ≥50%. Margetuximab was given by intravenous infusion at 0.1 – 6.0 mg/kg for 3 of every 4 weeks or once every 3 weeks (10 – 18 mg/kg). Results Three of 17 HER2 3+ metastatic breast cancer patients received long-term margetuximab. Patient 35 had 3 prior regimens (adjuvant doxorubicin+cyclophosphamide followed by docetaxol+trastuzumab; gemcitabine+vinorelbine; lapatinib+capecitabine) and received margetuximab at 10 mg/kg q3wk, 88 cycles to date, with PR achieved Cycle 1 Day 43, maintained 4.4 years. Patient 44 had 3 prior regimens for metastatic disease (docetaxel+trastuzumab+pertuzumab; doxorubicin+cyclophosphamide; lapatinib+capecitabine) and received margetuximab at 15 mg/kg q3wk, 79 cycles to date with SD for 4.3 years. Patient 50 had 4 prior regimens for recurrent/metastatic disease (tamoxifen; anastrozole; capecitabine+trastuzumab; lapatinib+capecitabine) and received margetuximab dose of 18 mg/kg q3wk with PR achieved Cycle 1 Day 43, maintained 3.5 years. Progression was noted at Cycle 57, and margetuximab continues at 63 cycles to date. No cardiac toxicities were found during long-term follow-up for these 3 patients and there were no treatment-related adverse events ≥Grade 3. Conclusions Margetuximab is well-tolerated without cardiac toxicities in long-term responders, with single-agent activity including durable responses in heavily pre-treated metastatic breast cancer. A Phase 3, randomized, multi-center clinical trial (SOPHIA; NCT02492711) is enrolling patients with metastatic breast cancer, comparing margetuximab plus chemotherapy to trastuzumab plus chemotherapy in patients who have received 1 to 3 lines of therapy for advanced disease. Citation Format: Im S-A, Bang Y-J, Oh D-Y, Giaccone G, Bauer T, Nordstrom J, Li H, Moore P, Hong S, Baughman J, Rock E, Burris H. Long-term responders to single-agent margetuximab, an Fc-modified anti-HER2 monoclonal antibody, in metastatic HER2+ breast cancer patients with prior anti-HER2 therapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-18-11.

Aptamer-integrated α-Gal liposomes as bispecific agents to trigger immune response for killing tumor cells.

A novel bispecific α-Gal liposome was constructed by self-assembling AS1411 aptamers into the α-Gal containing liposomes. The α-Gal liposomes were prepared using cell membranes of red blood cells from rabbit, which are composed of cholesterol, phospholipids, and α-Gal glycolipids. AS1411 is a DNA aptamer with high specificity and affinity for nucleolin and could integrate into liposomes by the modification of cholesterol. The bispecific α-Gal liposomes surface-functionalized by α-Gal and AS1411 aptamer could recognize anti-Gal antibodies and nucleolin overexpressed by tumor cells simultaneously, followed by activating the immune system to attack the tumor cells, resulting in the lysis of the tumor cells by antibody dependent cell-mediated cytotoxicity. Under simulated tumor environment, the lysis rate of MCF-7 cells treated by the AS1411 modified α-Gal liposomes drastically increased compared to the liposomes without AS1411 aptamer. This study suggests that the AS1411 modified α-Gal liposomes can recognize nucleolin-overexpressing tumor cells selectively, subsequently improve the effect of the immunotherapy with high specificity. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1176-1183, 2019.