Antibody-dependent Cytotoxicity Research Articles

Anti-tumor activity of cetuximab plus avelumab in non-small cell lung cancer patients involves innate immunity activation: findings from the CAVE-Lung trial

BackgroundWe recently conducted Cetuximab-AVElumab-Lung (CAVE-Lung), a proof-of-concept, translational and clinical trial, to evaluate the combination of two IgG1 monoclonal antibodies (mAb): avelumab, an anti-PD-L1 drug, and cetuximab, an anti-epidermal growth factor receptor (EGFR) drug, as second- or third-line treatment in non-small cell lung cancer (NSCLC) patients. We have reported clinically relevant anti-tumor activity in 6/16 patients. Clinical benefit was accompanied by Natural Killer (NK) cell-mediated antibody-dependent cell cytotoxicity (ADCC). Among the 6 responding patients, 3 had progressed after initial response to a previous treatment with single agent anti-PD-1, nivolumab or pembrolizumab.MethodsWe report long-term clinical follow-up and additional findings on the anti-tumor activity and on the immune effects of cetuximab plus avelumab treatment for these 3 patients.ResultsAs of November 30, 2021, 2/3 patients were alive. One patient was still on treatment from 34 months, while the other two patients had progression free survival (PFS) of 15 and 19 months, respectively. Analysis of serially collected peripheral blood mononuclear cells (PBMC) revealed long-term activation of NK cell-mediated ADCC. Comprehensive genomic profile analysis found somatic mutations and germline rare variants in DNA damage response (DDR) genes. Furthermore, by transcriptomic analysis of The Cancer Genome Atlas (TCGA) dataset we found that DDR mutant NSCLC displayed high STING pathway gene expression. In NSCLC patient-derived three-dimensional in vitro spheroid cultures, cetuximab plus avelumab treatment induced additive cancer cell growth inhibition as compared to single agent treatment. This effect was partially blocked by treatment with an anti-CD16 mAb, suggesting a direct involvement of NK cell activation. Furthermore, cetuximab plus avelumab treatment induced 10-, 20-, and 20-fold increase, respectively, in the gene expression of CCL5 and CXCL10, two STING downstream effector cytokines, and of interferon β, as compared to untreated control samples.ConclusionsDDR mutations may contribute to DDR-induced STING pathway with sustained innate immunity activation following cetuximab plus avelumab combination in previously treated, PD-1 inhibitor responsive NSCLC patients.

Delayed B cell repopulation after rituximab treatment in multiple sclerosis patients with expanded adaptive natural killer cells.

Background and purposeThe aim was to evaluate whether adaptive NKG2C+ natural killer (NK) cells, characterized by enhanced antibody‐dependent cell cytotoxicity (ADCC), may influence time to B cell repopulation after rituximab treatment in multiple sclerosis (MS) patients.MethodsThis was a prospective observational study of MS patients treated with rituximab monitoring peripheral B cells for repeated doses. B cell repopulation was defined as CD19+ cells above 2% of total lymphocytes, classifying cases according to the median time of B cell repopulation as early or late (≤9 months, >9 months, respectively). Basal NK cell immunophenotype and in vitro ADCC responses induced by rituximab were assessed by flow cytometry.ResultsB cell repopulation in 38 patients (24 relapsing–remitting MS [RRMS]; 14 progressive MS) was classified as early (≤9 months, n = 19) or late (>9 months, n = 19). RRMS patients with late B cell repopulation had higher proportions of NKG2C+ NK cells compared to those with early repopulation (24.7% ± 16.2% vs. 11.3% ± 10.4%, p < 0.05), and a direct correlation between time to B cell repopulation and percentage of NKG2C+ NK cells (R 0.45, p < 0.05) was observed. RRMS cases with late repopulation compared with early repopulation had a higher secretion of tumor necrosis factor α and interferon γ by NK cells after rituximab‐dependent NK cell activation. The NK cell immunophenotype appeared unrelated to B cell repopulation in progressive MS patients.ConclusionsAdaptive NKG2C+ NK cells in RRMS may be associated with delayed B cell repopulation after rituximab, a finding probably related to enhanced depletion of B cells exerted by NK‐cell‐mediated ADCC, pointing to the use of personalized regimens with anti‐CD20 monoclonal antibody therapy in some patients.

MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

ABSTRACT The major histocompatibility complex-class I chain related proteins A and B (MICA/B) is upregulated because of cellular stress and MICA/B shedding by cancer cells causes escape from NKG2D recognition favoring the emergence of cancers. Cholangiocarcinoma (CCA) is a relatively rare, though increasingly prevalent, primary liver cancer characterized by a late clinical presentation and a dismal prognosis. We explored the NKG2D-MICA/B axis in NK cells from 41 patients with intrahepatic cholangiocarcinoma (iCCA). The MICA/B-specific 7C6 mAb was used for ex vivo antibody-dependent cytotoxicity (ADCC) experiments using circulating, non tumor liver- and tumor-infiltrating NK cells against the HuCCT-1 cell line and patient-derived primary iCCA cells as targets. MICA/B were more expressed in iCCA than in non-tumoral tissue, MICA transcription being higher in moderately-differentiated compared with poorly-differentiated cancer. Serum MICA was elevated in iCCA patients in line with higher expression of ADAM10 and ADAM17 that are responsible for proteolytic release of MICA/B from tumor. Addition of 7C6 significantly boosted peripheral, liver- and tumor-infiltrating-NK cell degranulation and IFNγ production toward MICA/B-expressing established cell lines and autologous iCCA patient target cells. Our data show that anti-MICA/B drives NK cell anti-tumor activity, and provide preclinical evidence in support of 7C6 as a potential immunotherapeutic tool for iCCA.

Molecular Aspects of Resistance to Immunotherapies-Advances in Understanding and Management of Diffuse Large B-Cell Lymphoma.

Despite the unquestionable success achieved by rituximab-based regimens in the management of diffuse large B-cell lymphoma (DLBCL), the high incidence of relapsed/refractory disease still remains a challenge. The widespread clinical use of chemo-immunotherapy demonstrated that it invariably leads to the induction of resistance; however, the molecular mechanisms underlying this phenomenon remain unclear. Rituximab-mediated therapeutic effect primarily relies on complement-dependent cytotoxicity and antibody-dependent cell cytotoxicity, and their outcome is often compromised following the development of resistance. Factors involved include inherent genetic characteristics and rituximab-induced changes in effectors cells, the role of ligand/receptor interactions between target and effector cells, and the tumor microenvironment. This review focuses on summarizing the emerging advances in the understanding of the molecular basis responsible for the resistance induced by various forms of immunotherapy used in DLBCL. We outline available models of resistance and delineate solutions that may improve the efficacy of standard therapeutic protocols, which might be essential for the rational design of novel therapeutic regimens.

Overview of Memory NK Cells in Viral Infections: Possible Role in SARS-CoV-2 Infection

NK cells have usually been defined as cells of the innate immune system, although they are also involved in adaptative responses. These cells belong to the innate lymphocyte cells (ILC) family. They remove unwanted cells, tumoral cells and pathogens. NK cells are essential for viral infection clearance and are involved in tolerogenic responses depending on the dynamic balance of the repertoire of activating and inhibitory receptors. NK plasticity is crucial for tissue function and vigilant immune responses. They directly eliminate virus-infected cells by recognising viral protein antigens using a non-MHC dependent mechanism, recognising viral glycan structures and antigens by NCR family receptors, inducing apoptosis by Fas-Fas ligand interaction, and killing cells by antibody-dependent cell cytotoxicity via the FcγIII receptor. Activating receptors are responsible for the clearance of virally infected cells, while inhibitory KIR receptor activation impairs NK responses and facilitates virus escape. Effective NK memory cells have been described and characterised by a low NKG2A and high NKG2C or NKG2D expression. NK cells have also been used in cell therapy. In SARS-CoV-2 infection, several contradicting reports about the role of NK cells have been published. A careful analysis of the current data and possible implications will be discussed.

Macrophage-derived CXCL9 and CXCL11, T-cell skin homing, and disease control in mogamulizumab-treated CTCL patients

Cutaneous T-cell lymphomas (CTCLs) are rare malignancies involving primarily the skin. Responses to treatment are usually short-lived in advanced CTCL. The determinants of long-term CTCL control are unclear. Mogamulizumab, an anti-human CCR4 antibody that acts by antibody-dependent cell cytotoxicity against CCR4+ CTCL tumor cells and peripheral memory blood regulatory T cells, has been associated with long-lasting remissions and immune adverse events. Here, we reported skin rashes in 32% of 44 patients with CTCL treated with mogamulizumab, associated with significantly higher overall survival (hazard ratio, 0.16; 0.04-0.73; P = .01). Rash occurred in patients with Sézary syndrome and was associated with longer time to progression. These rashes were characterized by a CD163+ granulomatous and/or CD8+ lichenoid skin infiltrate. High-throughput sequencing analysis of T-cell receptor β genes in skin and blood flow cytometry confirmed the depletion of CTCL tumor cells, as well as the recruitment of new reactive T-cell clones in skin at the time of skin rash. CXCL9 and CXCL11, two macrophage-derived chemokines that recruit CXCR3+ T cells to skin, were overexpressed in skin rashes. A higher frequency of TIGIT+ and PD1+ exhausted reactive blood T cells was observed at baseline in patients with rash, and this frequency decreased with mogamulizumab treatment. These data are consistent with mogamulizumab-induced long-term immune CTCL control by activation of the macrophage and T-cell responses in patients with rash.

CD137 Costimulation Counteracts TGFβ Inhibition of NK-cell Antitumor Function.

Enhancing natural killer (NK) cell-based cancer immunotherapy by overcoming immunosuppression is an area of intensive research. Here, we have demonstrated that the anti-CD137 agonist urelumab can overcome TGFβ-mediated inhibition of human NK-cell proliferation and antitumor function. Transcriptomic, immunophenotypic, and functional analyses showed that CD137 costimulation modified the transcriptional program induced by TGFβ on human NK cells by rescuing their proliferation in response to IL2, preserving their expression of activating receptors (NKG2D) and effector molecules (granzyme B, IFNγ) while allowing the acquisition of tumor-homing/retention features (CXCR3, CD103). Activated NK cells cultured in the presence of TGFβ1 and CD137 agonist recovered CCL5 and IFNγ secretion and showed enhanced direct and antibody-dependent cytotoxicity upon restimulation with cancer cells. Trastuzumab treatment of fresh breast carcinoma-derived multicellular cultures induced CD137 expression on tumor-infiltrating CD16+ NK cells, enabling the action of urelumab, which fostered tumor-infiltrating NK cells and recapitulated the enhancement of CCL5 and IFNγ production. Bioinformatic analysis pointed to IFNG as the driver of the association between NK cells and clinical response to trastuzumab in patients with HER2-positive primary breast cancer, highlighting the translational relevance of the CD137 costimulatory axis for enhancing IFNγ production. Our data reveals CD137 as a targetable checkpoint for overturning TGFβ constraints on NK-cell antitumor responses.

EXTH-02. AAV MEDIATED BRAIN DELIVERY OF AN ADCC-ENHANCED ANTIBODY OBVIATES XENOGRAFT GROWTH IN MOUSE MODELS OF HER2+ BREAST CANCER BRAIN METASTASIS

Abstract BACKGROUND HER2+ tumors constitute approximately 20% of breast cancer patients and are characterized by overexpression of the growth factor receptor HER2 (ERBB2), a cell proliferation driver. Effective anti-HER2 therapies confer prolonged patient survival necessitating the need for transformative treatments targeting brain metastases, a major cause of mortality in ~30-50% of HER2+ metastatic breast cancer patients. HER2-directed antibody immunotherapy, while efficacious for peripheral disease, has limited central nervous system exposure (CNS). To overcome these challenges, we transduced CNS cells with a novel AAV vector carrying an anti-HER2 antibody payload. METHODS We assessed the biochemical equivalence and functional effectiveness of AAV vector-encoded antibodies using in vitro assays. After selecting promising vector-encoded antibody candidates, a novel, blood-brain barrier penetrant AAV capsid was administered via i.v. dosing to an orthotopic xenograft mouse model of HER2+ brain metastases. Bioluminescent imaging provided a longitudinal measure of brain tumor burden. At study termination, we measured antibody biodistribution in cerebrospinal fluid (CSF), serum, and brain homogenates with AlphaLISA assays. RESULTS Using HER2+ breast cancer cell lines, we determined that an antibody-dependent cell cytotoxicity (ADCC) enhanced anti-HER2 antibody was most effective and demonstrated that AAV-vector encoded forms of the antibody performed comparably to recombinant reference antibodies. Following i.v. administration of a HER2 antibody encoding AAV vector, we measured &amp;gt;1 ug/mL of the antibody in CSF. Importantly, AAV-mediated expression of the ADCC-enhanced HER2-directed antibody significantly abrogated tumor growth in orthotopic xenograft models. CONCLUSIONS Peripheral administration of an AAV vector was able to transduce brain tissue such that efficacious levels of HER2-directed antibodies were produced. This strategy was successful at preventing tumor growth in our physiologically relevant model of breast cancer brain metastases. Such a treatment modality should be further evaluated in patient derived PDX models to validate translational efficacy for human patients.

Safety and Efficacy of FT596, a First-in-Class, Multi-Antigen Targeted, Off-the-Shelf, iPSC-Derived CD19 CAR NK Cell Therapy in Relapsed/Refractory B-Cell Lymphoma

Safety and Efficacy of FT596, a First-in-Class, Multi-Antigen Targeted, Off-the-Shelf, iPSC-Derived CD19 CAR NK Cell Therapy in Relapsed/Refractory B-Cell Lymphoma

Enhancing Natural Killer Cell Targeting of Pediatric Sarcoma

Osteosarcoma, Ewing sarcoma (EWS), and rhabdomyosarcoma (RMS) are the most common pediatric sarcomas. Conventional therapy for these sarcomas comprises neoadjuvant and adjuvant chemotherapy, surgical resection of the primary tumor and/or radiation therapy. Patients with metastatic, relapsed, or refractory tumors have a dismal prognosis due to resistance to these conventional therapies. Therefore, innovative therapeutic interventions, such as immunotherapy, are urgently needed. Recently, cancer research has focused attention on natural killer (NK) cells due their innate ability to recognize and kill tumor cells. Osteosarcoma, EWS and RMS, are known to be sensitive to NK cell cytotoxicity in vitro. In the clinical setting however, NK cell cytotoxicity against sarcoma cells has been mainly studied in the context of allogeneic stem cell transplantation, where a rapid immune reconstitution of NK cells plays a key role in the control of the disease, known as graft-versus-tumor effect. In this review, we discuss the evidence for the current and future strategies to enhance the NK cell-versus-pediatric sarcoma effect, with a clinical focus. The different approaches encompass enhancing antibody-dependent NK cell cytotoxicity, counteracting the NK cell mechanisms of self-tolerance, and developing adoptive NK cell therapy including chimeric antigen receptor-expressing NK cells.

Cellular and Molecular Crosstalk of Graft Endothelial Cells During AMR: Effector Functions and Mechanisms.

Graft endothelial cell (EC) injury is central to the pathogenesis of antibody-mediated rejection (AMR). The ability of donor-specific antibodies (DSA) to bind C1q and activate the classical complement pathway is an efficient predictor of graft rejection highlighting complement-dependent cytotoxicity as a key process operating during AMR. In the past 5 y, clinical studies further established the cellular and molecular signatures of AMR revealing the key contribution of other, IgG-dependent and -independent, effector mechanisms mediated by infiltrating NK cells and macrophages. Beyond binding to alloantigens, DSA IgG can activate NK cells and mediate antibody-dependent cell cytotoxicity through interacting with Fcγ receptors (FcγRs) such as FcγRIIIa (CD16a). FcRn, a nonconventional FcγR that allows IgG recycling, is highly expressed on ECs and may contribute to the long-term persistence of DSA in blood. Activation of NK cells and macrophages results in the production of proinflammatory cytokines such as TNF and IFNγ that induce transient and reversible changes in the EC phenotype and functions promoting coagulation, inflammation, vascular permeability, leukocyte trafficking. MHC class I mismatch between transplant donor and recipient can create a situation of "missing self" allowing NK cells to kill graft ECs. Depending on the microenvironment, cellular proximity with ECs may participate in macrophage polarization toward an M1 proinflammatory or an M2 phenotype favoring inflammation or vascular repair. Monocytes/macrophages participate in the loss of endothelial specificity in the process of endothelial-to-mesenchymal transition involved in renal and cardiac fibrosis and AMR and may differentiate into ECs enabling vessel and graft (re)-endothelialization.

Profile of Immunoglobulin G N-Glycome in COVID-19 Patients: A Case-Control Study.

Coronavirus disease 2019 (COVID-19) remains a major health challenge globally. Previous studies have suggested that changes in the glycosylation of IgG are closely associated with the severity of COVID-19. This study aimed to compare the profiles of IgG N-glycome between COVID-19 patients and healthy controls. A case-control study was conducted, in which 104 COVID-19 patients and 104 age- and sex-matched healthy individuals were recruited. Serum IgG N-glycome composition was analyzed by hydrophilic interaction liquid chromatography with the ultra-high-performance liquid chromatography (HILIC-UPLC) approach. COVID-19 patients have a decreased level of IgG fucosylation, which upregulates antibody-dependent cell cytotoxicity (ADCC) in acute immune responses. In severe cases, a low level of IgG sialylation contributes to the ADCC-regulated enhancement of inflammatory cytokines. The decreases in sialylation and galactosylation play a role in COVID-19 pathogenesis via the activation of the lectin-initiated alternative complement pathway. IgG N-glycosylation underlines the complex clinical phenotypes of SARS-CoV-2 infection.

BL-01, an Fc-bearing, tetravalent CD20 × CD5 bispecific antibody, redirects multiple immune cells to kill tumors in vitro and in vivo

Background aimsThe authors describe here a novel therapeutic strategy combining a bispecific antibody (bsAb) with cytokine-induced killer (CIK) cells. MethodsThe authors have designed, produced and purified a novel tetravalent IgG1-like CD20 × CD5 bsAb called BL-01. The bsAb is composed of a fused heavy chain and two free light chains that pair correctly to the heavy chain sequences thanks to complementary mutations in the monoclonal antibody 2 CH1/CL sequences. ResultsThe authors show that BL-01 can bind specifically to CD20 and CD5 with an affinity of 4–6 nM, demonstrating correct pairing of two light chains to the fused heavy chain. The CD20 × CD5 BL-01 bsAb has a functional human IgG1 Fc and can induce up to 65% complement-dependent cytotoxicity of a CD20+ lymphoma cell line in the presence of human complement, similar to anti-CD20 rituximab. The bsAb also induces significant natural killer cell activation and antibody-dependent cytotoxicity of up to 25% as well as up to 65% phagocytosis by human macrophages in the presence of CD20+ tumor cells. The BL-01 bsAb binds to CD20 and CD5 simultaneously and can redirect CIK cells in vitro to kill CD20+ targets, increasing the cytotoxicity of CIK cells by about 3-fold. The authors finally show that the CD20 × CD5 BL-01 bsAb synergizes with CIK cells in vivo in controlling tumor growth and prolonging survival of nonobese diabetic/severe combined immunodeficiency mice inoculated with a patient-derived, aggressive diffuse large B-cell lymphoma xenograft. ConclusionsThe authors suggest that the efficacy of bsAb in vivo is due to the combined activation of innate immunity by Fc and redirection of CIK cells to kill the tumor target.

Exploiting B Cell Transfer for Cancer Therapy: Engineered B Cells to Eradicate Tumors.

Nowadays, cancers still represent a significant health burden, accounting for around 10 million deaths per year, due to ageing populations and inefficient treatments for some refractory cancers. Immunotherapy strategies that modulate the patient’s immune system have emerged as good treatment options. Among them, the adoptive transfer of B cells selected ex vivo showed promising results, with a reduction in tumor growth in several cancer mouse models, often associated with antitumoral immune responses. Aside from the benefits of their intrinsic properties, including antigen presentation, antibody secretion, homing and long-term persistence, B cells can be modified prior to reinfusion to increase their therapeutic role. For instance, B cells have been modified mainly to boost their immuno-stimulatory activation potential by forcing the expression of costimulatory ligands using defined culture conditions or gene insertion. Moreover, tumor-specific antigen presentation by infused B cells has been increased by ex vivo antigen loading (peptides, RNA, DNA, virus) or by the sorting/ engineering of B cells with a B cell receptor specific to tumor antigens. Editing of the BCR also rewires B cell specificity toward tumor antigens, and may trigger, upon antigen recognition, the secretion of antitumor antibodies by differentiated plasma cells that can then be recognized by other immune components or cells involved in tumor clearance by antibody-dependent cell cytotoxicity or complement-dependent cytotoxicity for example. With the expansion of gene editing methodologies, new strategies to reprogram immune cells with whole synthetic circuits are being explored: modified B cells can sense disease-specific biomarkers and, in response, trigger the expression of therapeutic molecules, such as molecules that counteract the tumoral immunosuppressive microenvironment. Such strategies remain in their infancy for implementation in B cells, but are likely to expand in the coming years.

Development of a novel anti-B7-H4 antibody enhances anti-tumor immune response of human T cells

BackgroundB7-H4 is a member of the B7 superfamily that is expressed on the surface of tumors and exhibits limited expression on normal tissue. B7-H4 negatively regulates tumor immunity by interacting with the B7-H4 receptor, which is expressed by activated CD8 + T cells. Hence, we sought to generate an immunomodulatory antibody that targets B7-H4 and blocks the immunosuppressive activity of B7-H4. MethodsAnti-B7-H4 antibodies were generated using the hybridoma technique and screened by a binding assay based on B7-H4-expressing tumor cells. The B7-H4 antagonistic antibodies were further screened based on their checkpoint blockade activity using a SEB-stimulated peripheral blood mononuclear cell (PBMC) assay, which comprised B7-H4-expressing antigen presenting cells (APCs) and activated T cells. To assess the immunomodulatory activity of anti-B7-H4 antibodies, activated human CD8+ T cells were cultured in B7-H4 protein-coated plates, and the production of IL-2 and the proliferation rate of CD8+ T cells were measured. In addition, we evaluated the ADCC effect of anti-B7-H4 antibodies against tumor cell lines. The in vivo antitumor efficacy of the anti-B7-H4 antibody was also evaluated in human T cell-engrafted NOG mice. ResultsA panel of anti-B7-H4 antibodies was generated. The top 23 antibodies were screened to identify antibodies that disabled B7-H4-mediated inhibition. Antibody 17 exhibited the greatest induction of the production of IL-2 and IFN-gamma in SEB-stimulated PBMCs. Antibody 17 was constructed as a chimeric antibody (CH17) with a human IgG1 constant domain. CH17 showed high affinity for human B7-H4 and fully cross-reacted with cynomolgus B7-H4. Additionally, CH17 mediated potent antibody-dependent cell cytotoxicity (ADCC) against different B7-H4-positive tumor cell lines. More importantly, CH17 relieved B7-H4-mediated T cell suppression by enhancing IL2 production and promoting T cell proliferation. In an MDA-MB-468-bearing mouse model in which human pan-T cells were engrafted, CH17 delayed tumor growth by engaging T cells and exerted a synergistic effect in combination with an anti-human PD-1 antibody. ConclusionsWe successfully generated an immunomodulatory antibody targeting B7-H4 that possesses both T cell immune checkpoint inhibitory activity and ADCC activity in B7-H4-positive tumors. B7-H4-targeting antibodies might represent a promising immunotherapy for B7-H4-expressing tumors.

HLA Class I Molecules as Immune Checkpoints for NK Cell Alloreactivity and Anti-Viral Immunity in Kidney Transplantation.

Natural killer (NK) cells are innate lymphocytes that can kill diseased- or virally-infected cells, mediate antibody dependent cytotoxicity and produce type I immune-associated cytokines upon activation. NK cells also contribute to the allo-immune response upon kidney transplantation either by promoting allograft rejection through lysis of cells of the transplanted organ or by promoting alloreactive T cells. In addition, they protect against viral infections upon transplantation which may be especially relevant in patients receiving high dose immune suppression. NK cell activation is tightly regulated through the integrated balance of signaling via inhibitory- and activating receptors. HLA class I molecules are critical regulators of NK cell activation through the interaction with inhibitory- as well as activating NK cell receptors, hence, HLA molecules act as critical immune checkpoints for NK cells. In the current review, we evaluate how NK cell alloreactivity and anti-viral immunity are regulated by NK cell receptors belonging to the KIR family and interacting with classical HLA class I molecules, or by NKG2A/C and LILRB1/KIR2DL4 engaging non-classical HLA-E or -G. In addition, we provide an overview of the methods to determine genetic variation in these receptors and their HLA ligands.

Development of Bacteriophage Virus-Like Particle Vaccines Displaying Conserved Epitopes of Dengue Virus Non-Structural Protein 1.

Dengue virus (DENV) is a major global health problem, with over half of the world’s population at risk of infection. Despite over 60 years of efforts, no licensed vaccine suitable for population-based immunization against DENV is available. Here, we describe efforts to engineer epitope-based vaccines against DENV non-structural protein 1 (NS1). NS1 is present in DENV-infected cells as well as secreted into the blood of infected individuals. NS1 causes disruption of endothelial cell barriers, resulting in plasma leakage and hemorrhage. Immunizing against NS1 could elicit antibodies that block NS1 function and also target NS1-infected cells for antibody-dependent cell cytotoxicity. We identified highly conserved regions of NS1 from all four DENV serotypes. We generated synthetic peptides to these regions and chemically conjugated them to bacteriophage Qβ virus-like particles (VLPs). Mice were immunized two times with the candidate vaccines and sera were tested for the presence of antibodies that bound to the cognate peptide, recombinant NS1 from all four DENV serotypes, and DENV-2-infected cells. We found that two of the candidate vaccines elicited antibodies that bound to recombinant NS1, and one candidate vaccine elicited antibodies that bound to DENV-infected cells. These results show that an epitope-specific vaccine against conserved regions of NS1 could be a promising approach for DENV vaccines or therapeutics to bind circulating NS1 protein.

Abstract 1306: Primary NK cells and luciferase expressing reporter cell lines for use in developing ADCC assays for immuno-oncology drug screening

Abstract Immuno-oncology is one of the fastest growing fields in oncology, and cancer immunotherapy in recent years represents a milestone in the treatment of cancer. Natural killer (NK) cells are potent innate effectors capable of targeting and killing virally infected and malignant cells. The use of monoclonal antibodies (mAbs) targeting tumor antigens triggers antibody-dependent cellular cytotoxicity (ADCC) by NK cells, which has shown prominent clinical efficacy in clearing tumors in cancer patients. Therefore, the development of tumor cell targeting mAbs and biologics dictate a clear need for human cell-based models to evaluate antibody efficacy. In this study, human PBMCs and purified NK cells from healthy donors are used to develop an ADCC assay. The NK cells purified from PBMCs are &amp;gt;90% CD45+ CD56+, express high levels of CD16, and NK activation markers such NKG2A, KIR2DL1, NKp44 upon activation. The activated NK cells exhibit both strong direct and antibody-dependent cytotoxicity. Genotyping for FcγRIIIa receptor F and V variant is also performed for cells isolated from different donors, which is the genetic polymorphism that affects ADCC response. In addition, we employ a portfolio of luciferase reporter tumor cell lines to evaluate NK cell direct killing and ADCC targeted killing. These include K562, Wil2S, BT474, A431, A375 parental, and CRISPR edited A375 KRAS mutant cell lines engineered to express luc2 reporter. The commonly targeted cell surface markers, such as CD20, Her2, and EGFR, are highly expressed within this panel of target tumor cell lines. We validate the ADCC assay system through testing the efficacy for some of the well-known mAb biologics such as anti-CD20, anti-HER2, and anti-EGFR. After co-culture of activated primary NK cells with luciferase reporter target cell lines and the addition of dilutions of ADCC and isotype control antibodies, we are able to show target cell specific and dose-dependent killing effects. The luciferase reporter provides great convenience in measuring target cell killing without having to use radioactive 51Cr release assay or pre-label the cells with calcein. This data demonstrates the utility of primary NK cells along with a portfolio of well characterized luciferase reporter tumor cell line to develop “custom” designed ADCC assays for studying NK cell immune response and screening for biological drugs for cancer immunotherapy. Citation Format: Haiyun Liu, Brian Della Fera, John Foulke, Luping Chen, Fang Tian. Primary NK cells and luciferase expressing reporter cell lines for use in developing ADCC assays for immuno-oncology drug screening [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 1306.

Abstract 1833: Development of anti-CLDN18.2-anti-CD3 bispecific antibody as cancer therapeutics

Abstract Claudin-18 (CLDN18) is a member of the large Claudin family of four-span transmembrane proteins, which are essential components of the mammalian tight junctions (TJs) in the epithelial cells. Claudin-18 has two splice variants, CLDN18.1 and CLDN18.2. While CLDN18.1 is specifically expressed in the lung tissue, CLDN18.2 expression in normal tissue is more restricted and is only detected in small patches of stomach mucosal. CLDN18.2 expression is elevated in many types of epithelial cancers including stomach, esophagus, pancreatic and ovarian cancers. Anti-CLDN18.2 monoclonal antibody Claudiximab (IMAB362) demonstrated promising results in clinical trials for gastric cancers. Using a combination of DNA, protein, and transfected cells as immunogens, we have generated a panel of mouse monoclonal antibodies which are highly specific for CLDN18.2 but do not cross-react to CLDN18.1. The lead antibody E9 had higher binding affinity for CLDN18.2, compared to the IMAB362. The humanized anti-CLDN18.2 antibody showed a strong antibody dependent cytotoxicity (ADCC) activity against Mia PaCa-2 cells overexpressing CLDN18.2. To redirect T-cell-mediated lysis toward CLDN18.2-overexpressed tumor cells, we generated an anti-CLDN18.2/anti-CD3 bispecific antibody. We demonstrated that the bispecific antibody binds to both human T cells and CLDN18.2-expressing tumor cells. Furthermore, the anti-CLDN18.2/anti-CD3 bispecific antibody had robust activity in killing Mia PaCa-2 cells overexpressing CLDN18.2 in the in vitro T cell-dependent cellular cytotoxicity (TDCC) assay. In addition, the bispecific antibody retained the in vitro ADCC activity against Mia PaCa-2 cells overexpressing CLDN18.2. Taken together, our data suggest that the anti-CLDN18.2/anti-CD3 bispecific antibody is a promising cancer therapeutic candidate utilizing multiple anti-tumor mechanisms of action, including ADCC and TDCC. Citation Format: Haishan Lin, Huey Li, Hunter Drobenaire, Veronica Pizzarella, Richard Zhang. Development of anti-CLDN18.2-anti-CD3 bispecific antibody as cancer therapeutics [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 1833.

PD-L1 targeting and subclonal immune escape mediated by PD-L1 mutations in metastatic colorectal cancer

BackgroundIn patients with microsatellite stable (MSS) metastatic colorectal cancer (mCRC), immune checkpoint blockade is ineffective, and combinatorial approaches enhancing immunogenicity need exploration.MethodsWe treated 43 patients with predominantly microsatellite stable RAS/BRAF...