Antibody-dependent Cytotoxicity Research Articles

Memory-like differentiation enhances NK cell responses against colorectal cancer

ABSTRACT Metastatic (m) colorectal cancer (CRC) is an incurable disease with a poor prognosis and thus remains an unmet clinical need. Immune checkpoint blockade (ICB)-based immunotherapy is effective for mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) mCRC patients, but it does not benefit the majority of mCRC patients. NK cells are innate lymphoid cells with potent effector responses against a variety of tumor cells but are frequently dysfunctional in cancer patients. Memory-like (ML) NK cells differentiated after IL-12/IL-15/IL-18 activation overcome many challenges to effective NK cell anti-tumor responses, exhibiting enhanced recognition, function, and in vivo persistence. We hypothesized that ML differentiation enhances the NK cell responses to CRC. Compared to conventional (c) NK cells, ML NK cells displayed increased IFN-γ production against both CRC cell lines and primary patient-derived CRC spheroids. ML NK cells also exhibited improved killing of CRC target cells in vitro in short-term and sustained cytotoxicity assays, as well as in vivo in NSG mice. Mechanistically, enhanced ML NK cell responses were dependent on the activating receptor NKG2D as its blockade significantly decreased ML NK cell functions. Compared to cNK cells, ML NK cells exhibited greater antibody-dependent cytotoxicity when targeted against CRC by cetuximab. ML NK cells from healthy donors and mCRC patients exhibited increased anti-CRC responses. Collectively, our findings demonstrate that ML NK cells exhibit enhanced responses against CRC targets, warranting further investigation in clinical trials for mCRC patients, including those who have failed ICB.

Single shot dendritic cell targeting SARS-CoV-2 vaccine candidate induces broad, durable and protective systemic and mucosal immune responses in mice

Current COVID-19 vaccines face limitations including waning immunity, immune escape by SARS-CoV-2 variants, limited cellular response, and poor mucosal immunity. We engineered a Clec9A-Receptor Binding Domain (RBD) antibody construct that delivers the SARS-CoV-2 RBD to conventional type 1 dendritic cells. Compared to non-targeting approaches, single dose immunization in mice with Clec9A-RBD induced far higher RBD-specific antibody titers that were sustained for up to 21 months post-vaccination. Uniquely, increasing neutralizing and antibody-dependent cytotoxicity activities across the sarbecovirus family was observed, suggesting antibody affinity maturation over time. Consistently and remarkably, RBD-specific follicular T-helper cells and germinal center B cells persisted up to 12 months post-immunization. Furthermore, Clec9A-RBD immunization induced a durable mono- and poly-functional TH1-biased cellular response that was strongly cross-reactive against SARS-CoV-2 variants of concern, including Omicron subvariants, and with robust CD8+ T cell signature. Uniquely, Clec9A-RBD single-shot systemic immunization effectively primed RBD-specific cellular and humoral immunity in lung and resulted in significant protection against homologous SARS-CoV-2 challenge as evidenced by limited body weight loss and approx. 2 log10 reduction in lung viral loads compared to non-immunized controls. Therefore, Clec9A-RBD immunization has the potential to trigger robust and sustained, systemic and mucosal protective immunity against rapidly evolving SARS-CoV2 variants.

Distinct CD16a features on human NK cells observed by flow cytometry correlate with increased ADCC

Natural killer (NK) cells destroy tissue that have been opsonized with antibodies. Strategies to generate or identify cells with increased potency are expected to enhance NK cell-based immunotherapies. We previously generated NK cells with increased antibody-dependent cell mediated cytotoxicity (ADCC) following treatment with kifunensine, an inhibitor targeting mannosidases early in the N-glycan processing pathway. Kifunensine treatment also increased the antibody-binding affinity of Fc γ receptor IIIa/CD16a. Here we demonstrate that inhibiting NK cell N-glycan processing increased ADCC. We reduced N-glycan processing with the CRIPSR-CAS9 knockdown of MGAT1, another early-stage N-glycan processing enzyme, and showed that these cells likewise increased antibody binding affinity and ADCC. These experiments led to the observation that NK cells with diminished N-glycan processing capability also revealed a clear phenotype in flow cytometry experiments using the B73.1 and 3G8 antibodies binding two distinct CD16a epitopes. We evaluated this “affinity profiling” approach using primary NK cells and identified a distinct shift and differentiated populations by flow cytometry that correlated with increased ADCC.

Xianling Lianxia formula improves the efficacy of trastuzumab by enhancing NK cell-mediated ADCC in HER2-positive BC

Trastuzumab has improved survival rates in human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC), but drug resistance leads to treatment failure. Natural killer (NK) cell-mediated antibody-dependent cell cytotoxicity (ADCC) represents an essential antitumor immune mechanism of trastuzumab. Traditional Chinese medicine (TCM) has been used for centuries to treat diseases because of its capacity to improve immune responses. Xianling Lianxia formula (XLLXF), based on the principle of “strengthening body and eliminating toxin”, exhibits a synergistic effect in the trastuzumab treatment of patients with HER2-positive BC. Notably, this synergistic effect of XLLXF was executed by enhancing NK cells and ADCC, as demonstrated through in vitro co-culture of NK cells and BC cells and in vivo intervention experiments. Mechanistically, the augmented impact of XLLXF on NK cells is linked to a decrease in cytokine inducible SH2 containing protein (CISH) expression, which in turn activates the Janus kinase 1 (JAK1)/signal transducer and activator of transcription 5 (STAT5) pathway. Collectively, these findings suggested that XLLXF holds promise for enhancing NK cell function and sensitizing patients with HER2-positive BC to trastuzumab.

Strategic development of a self-adjuvanting SARS-CoV-2 RBD vaccine: From adjuvant screening to enhanced immunogenicity with a modified TLR7 agonist

Adjuvants enhance the body's immune response to a vaccine, often leading to better protection against diseases. Monophosphoryl lipid A analogues (MPLA, TLR4 agonists), α-galactosylceramide analogues (NKT cell agonists), and imidazoquinoline compounds (TLR7/8 agonists) are emerging novel adjuvants on market or under clinical trials. Despite significant interest in these adjuvants, a direct comparison of their adjuvant activities remains unexplored. We initially assessed the activities of various adjuvants from three distinct categories using the SARS-CoV-2 RBD trimer antigen. TLR4 and TLR7/8 agonists are discovered to elicit robust IgG2a/2b antibodies, which is crucial for eliciting antibody dependent cytotoxicity. While α-galactosylceramide analogs induced mainly IgG1 antibody. Then, because of the flexibility of the TLR7/8 agonist, we designed and synthesized a tri-component self-adjuvanting SARS-CoV-2 RBD vaccine, featuring a covalent TLR7 agonist and targeting mannoside. Animal studies indicated that this vaccine generated antigen-specific humoral immunity. Yet, its immunogenicity seems compromised, indicating the complexity of the vaccine.

Abstract 2736: Ligand-independent targeting of tumor cells for antibody-dependent cell-mediated cytotoxicity

Abstract Introduction: Biological targeting is a key concept in cancer treatment utilized by most therapeutics. Targeted agents that selectively treat diseased tissues while reducing off-target effects to normal tissues are widely used for cancer therapies. However, key hurdles include: (i) a limited number of tumor-specific biomarkers expressed in sufficiently high levels for targeted therapy, (ii) lack of specific biomarkers amenable to targeting, and (iii) current technologies rely on largely protein-based targeting ligands limiting the applications of these therapies due to final conjugates with low payloads. We developed a novel method for tumor-specific delivery by combining metabolic glycoengineering and nanomedicine to engineer targetable chemical groups on cancer cells and enable directed treatments. We hypothesize that increased Fc fragments on the surface of tumor cells can enable antibody-dependent cell mediated cytotoxicity (ADCC) response to tumor cells, providing innate immunotherapy. Materials/Methods: We formulated nanoparticles (NPs) using polyethylene glycol-ply lactic acid-co-glycolic acid (PEG-PLGA). Using the Dolomite microfluidic device, we formed NPs by nanoprecipitation of PEG-PLGA with tetraacetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz), a sialic acid analogue containing a click chemistry group. We targeted this reactive group by functionalizing the Fc fragment with dibenzylcyclooctyne (DBCO). We characterized the NPs using dynamic light scattering (DLS), charge measurements and transmission electron microscopy (TEM). We evaluated toxicity using a cell viability assay and assessed preliminary efficacy with a tumor growth delay study. Results: NP characterization revealed a size of 30-40 nm via DLS, congruent with TEM imaging. Charge measurements ranged from -4 to +4 mV. Toxicity studies revealed no differences in cell viability amongst different treatment groups. Preliminary efficacy studies demonstrated improved tumor growth delay in the cohort of mice treated with a combination of NPs and Fc-DBCO conjugate, as well as a survival benefit. Median OS was 31.5 days for the control groups vs 57.5 days for the combination treated group (p = 0.0067). Conclusion: There is growing interest in harnessing the power of the innate immune response to shape anti-tumor immunity. This is because clinical data demonstrates tumor cells can evade the adaptive immune system through mutations as well as downregulate MHC receptors. One of the most effective innate immune responses is ADCC. To this end, we developed a novel method for tumor-specific delivery of Fc fragments to enhance ADCC. While this data reveals improved tumor growth delay and a survival benefit, the sample size is limited, and a larger study is needed to evaluate the potential benefits of this strategy. Furthermore, correlative studies are needed to identify the mechanism for treatment response. Citation Format: Maureen L. Aliru, Nikhila R. Sultanpuram, Kin Man Au, Tian Zhang, Andrew Z. Wang. Ligand-independent targeting of tumor cells for antibody-dependent cell-mediated cytotoxicity [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 2736.

Abstract 5341: Novel FcγR humanized mouse model enables assessment of PK/PD of therapeutic antibodies

Abstract Therapeutic antibodies have revolutionized the way we treat cancer. Enhanced activity of therapeutic IgG can be achieved by the modulation of Fc binding to Fcγ receptors (FcγR), which will consequently modulate the Fc-effector functions triggered upon crosslinking of target and effector cells by these antibodies. Assessment of therapeutic antibodies pharmacodynamic (PD) in preclinical models is challenging, as the FcγR are different from mice to human, as well as their expression pattern. FcRn is involved in the recycling and transport of IgG and is a key determinant of the pharmacokinetics (PK) of IgG. We report here a novel mouse model expressing human FcγR with a human-like expression pattern, in which human FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB are expressed and replace their mouse orthologs. The model was intercrossed with the hSA/hFcRn mouse model (Viuff et al., 2016), which has shown to improve translatability of PK assessment of therapeutics with extended half-life via FcRn recycling and/or HSA binding. Investigation of FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB expression on DC, monocytes, granulocytes, NK, T and B cells shows that expression pattern in hFcγR mice is consistent with their expression on human PBMC, with a few exceptions. These receptors are functional, as a human IgG1 targeting mouse CD20 induces approximately 50% of B-cell depletion in blood and liver, after a single iv administration in hFcγR mice. This depletion is Fc-mediated, as it is abrogated when hFcγR mice are treated with an anti-mouse CD20 human IgG1 with a Fc-null mutation. Assessment of antibody-dependent cell cytotoxicity (ADCC) ex vivo to investigate FcγR functionality in NK cells shows that Rituximab, an anti-human CD20 chimeric IgG1 with regular Fc, induces higher ADCC efficacy in NK cells from hFcγR than in NK cells from WT mice. Obinutuzumab, an anti-human CD20 featuring an optimized Fc portion to enhance binding to FcγRIII, shows higher tumor cell lysis than Rituximab in NK cells from hFcγR mice, suggesting this model enables ranking of antibodies. Moreover, assessment of activity of Fc-engineered anti-influenza IgG antibodies also shows that anti-Flu antibody with enhanced Fc binding outperforms the WT antibody, improves protective activity, and demonstrates effector function in influenza challenge model, following prophylactic treatment. Conversely, hFcγR mice treated with an Fc-null version of the anti-Flu antibody shows reduced protective activity and survival compared with the WT antibody. Therefore, the model reported here is a novel tool for testing efficacy of Fc-engineered therapeutic antibodies. We are currently investigating the PK profile of therapeutic antibodies in the hFcγR/hSA/hFcRn model. This novel tool is being improved to enable tolerability to human IgG1, biodistribution to the central nervous system, and flexibility of test of therapeutics through expression of human immune checkpoints. Citation Format: Fabiane Sonego, Angela Pappalardo, Gaëlle Martin, Rachel Courtois, Séverine Augier, Yacine Cherifi, Ariane Morel, Patricia Isnard-Petit, Kader Thiam. Novel FcγR humanized mouse model enables assessment of PK/PD of therapeutic antibodies [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 5341.

Abstract 4085: The anti-VISTA x MSLN bispecific antibody-interferon beta mutein fusion protein: A trispecific immunocytokine with potent therapeutic efficacy against malignant pleural mesothelioma

Abstract Background Malignant pleural mesothelioma (MPM) is a rare and highly fatal cancer primarily induced by asbestos exposure. The 5-year overall survival rate for MPM patients is less than 20%. Before the FDA approval of the combination of immune checkpoint inhibitors (Nivolumab + Ipilimumab, CHECKMATE-743), chemotherapy was the only systemic therapy for MPM. However, the predominant histological type of MPM, epithelioid MPM, comprising approximately 70% of cases, showed limited clinical benefits from immune checkpoint inhibitors when compared to non-epithelioid histology. Consequently, there is an unmet medical need to develop a new anti-cancer drug specifically for the treatment of epithelioid MPM. In this study, we discovered that V-domain Ig suppressor of T cell activation (VISTA), an immune checkpoint protein, and Mesothelin (MSLN), a tumor differentiation antigen, are co-expressed in epithelioid MPM. Subsequently, we developed a biomarker-based anti-VISTA x MSLN bispecific antibody (BsAb) and anti-VISTA x MSLN BsAb-IFN-β mutein fusion protein (trispecific immunocytokine) for the treatment of epithelioid MPM. Method We designed and screened various structures of the anti-VISTA x MSLN BsAb. To produce the trispecific immunocytokine, we fused IFN-β mutein (R27T/C17S), which exhibits improved stability, productivity, and pharmacokinetic properties compare to recombinant IFN-β. The in vitro efficacy of the trispecific immunocytokine was tested in human MPM cell lines, and its in vivo efficacy was evaluated in a human MPM xenograft mouse model and a human IFNAR1/2 Knock-in (KI) mouse model. Results We demonstrated a strong positive correlation between the expression levels of VISTA and MSLN in epithelioid MPM. The Anti-VISTA x MSLN BsAb showed improved binding affinity and antibody-dependent cell cytotoxicity (ADCC) activity. The trispecific immunocytokine displays analogous biological activities to both Anti-VISTA x MSLN BsAb and IFN-β mutein. The trispecific immunocytokine exhibited potent in vivo and in vitro anti-cancer activities against epithelioid MPM preclinical models, operating through both direct cytotoxicity and indirect activation of immune cells. Conclusion The trispecific immunocytokine demonstrated robust in vitro and in vivo anti-cancer efficacy in the epithelioid MPM model. Our findings suggest that the trispecific immunocytoine is a promising drug candidate for epithelioid MPM. Citation Format: Hee Geon Park, Hyeon Ju Kim, Myeung Ryun Seo, Ji Eun Park, Tae Won Kim, Jeung Hoo Choi, Jay Park, Kyoung Song, Chan Gyu Lee, Hae Min Jeong, Jun Young Choi, Yeong Jeong Jeon, Yoon La Choi, Sung Youl Hong, Young Kee Shin. The anti-VISTA x MSLN bispecific antibody-interferon beta mutein fusion protein: A trispecific immunocytokine with potent therapeutic efficacy against malignant pleural mesothelioma [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 4085.

Abstract 2737: LIS22, a first in class polyclonal antibody immunotherapy in T cell blood cancers

Abstract Background: Several approaches such as antibody drug conjugate (ADC), chimeric antigen receptor T cells (CAR-T) and recently bispecific antibodies are being introduced as innovative weapon in B-Cell lymphoma treatment. In the opposite, very limited therapeutic options are available for the treatment of rare refractory T cell malignancies such as PTCL. The 5-year survival rate of PTCL is lower than 30% according to the recent clinical data. Developing new effective treatment strategies for PTCL is an urgent problem. LIS22, is a first in class glyco-humanized polyclonal antibody (GH-pAb), targeting multiple tumor-associated antigens simultaneously. In this study, we characterized the safety and efficacy of LIS22 in preclinical models of T cell malignancies. Material and Methods: LIS22 ability to induce Antibody-Dependent Cell Cytotoxicity (ADCC), Antibody-Dependent Cellular Phagocytosis (ADCP), Complement Dependent Cytotoxicity (CDC), and apoptosis was tested in a panel of hematologic malignancy cell lines and peripheral blood mononuclear cell (PBMC). To assess the targeting and recognition of LIS22 in PTCL patients, we evaluated the immunolabelling of LIS22 on patients’ biopsies (n=119) using tissue microarray. LIS22 efficacy in vivo were evaluated in NMRI nude mice and SRG rats using T1301 and Jurkat cancer cell lines. Pharmacokinetics and safety of this drug were assessed in cynomolgus monkeys after single and repeated IV dosing up to 50mg/kg. Results: LIS22 acts via several mechanisms, at 30µg/ml, it induced cytotoxicity via CDC (in 70%), ADCP (in 49%), ADCC (in 41%) and apoptosis (in 30%) of HPB-ALL human T blood cancer cell line but not in PBMC. LIS22 showed a potent in vitro antitumor activity in a panel of hematologic malignancy cell lines, it induced specific tumor cell CDC (EC50=41.4±28.9µg/mL). In both CDC and apoptosis cytotoxicity assays, LIS22 displayed a significantly higher potency on T cell blood cancers and no toxicity on healthy blood cells compared to PTCL clinically active drug Alemtuzumab (Anti-CD52). It was able to kill up to 100% of the cancer cells without affecting PBMC. In immunolabeling assay, LIS22 demonstrated cross-reaction to PTCL patient biopsy (staining up to 93%). In vivo efficacy studies in different mice and rat xenograft models, LIS22 induced a significant reduction of tumor growth up to 90% across several tested tumors. LIS22 was also characterized by high tolerance profile and satisfactory exposure in monkeys after repeated dosing (up to 250mg/kg of cumulative dose). Conclusion: LIS22 appeared as a novel and promising cancer immunotherapy against T Cell hematologic malignancies. It has already been administered to human in another indication and showed a satisfactory PK and safety profile (no signs of immunotoxicity or systemic cytokine release). LIS22 is now ready to enter Phase I/II in the coming months for the treatment of peripheral T cell lymphoma (PTCL). Citation Format: Carine Ciron, Francoise Shneiker, Ophélie Dauphouy, Juliette Rousse, Pierre-Joseph Royer, Gwenaelle Evanno, Odile Duvaux, Firas Bassissi. LIS22, a first in class polyclonal antibody immunotherapy in T cell blood cancers [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 2737.

Tethering of soluble immune effectors to mucin and chitin reflects a convergent and dynamic role in gut immunity.

The immune system employs soluble effectors to shape luminal spaces. Antibodies are soluble molecules that effect immunological responses, including neutralization, opsonization, antibody-dependent cytotoxicity and complement activation. These molecules are comprised of immunoglobulin (Ig) domains. The N-terminal Ig domains recognize antigen, and the C-terminal domains facilitate their elimination through phagocytosis (opsonization). A less-recognized function mediated by the C-terminal Ig domains of the IgG class of antibodies (Fc region) involves the formation of multiple low-affinity bonds with the mucus matrix. This association anchors the antibody molecule to the matrix to entrap potential pathogens. Even though invertebrates are not known to have antibodies, protochordates have a class of secreted molecules containing Ig domains that can bind bacteria and potentially serve a similar purpose. The VCBPs (V region-containing chitin-binding proteins) possess a C-terminal chitin-binding domain that helps tether them to chitin-rich mucus gels, mimicking the IgG-mediated Fc trapping of microbes in mucus. The broad functional similarity of these structurally divergent, Ig-containing, secreted effectors makes a case for a unique form of convergent evolution within chordates. This opinion essay highlights emerging evidence that divergent secreted immune effectors with Ig-like domains evolved to manage immune recognition at mucosal surfaces in strikingly similar ways. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

Antibody dependent cellular cytotoxicity-inducing anti-EGFR antibodies as effective therapeutic option for cutaneous melanoma resistant to BRAF inhibitors.

About 50% of cutaneous melanoma (CM) patients present activating BRAF mutations that can be effectively targeted by BRAF inhibitors (BRAFi). However, 20% of CM patients exhibit intrinsic drug resistance to BRAFi, while most of the others develop adaptive resistance over time. The mechanisms involved in BRAFi resistance are disparate and globally seem to rewire the cellular signaling profile by up-regulating different receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR). RTKs inhibitors have not clearly demonstrated anti-tumor activity in BRAFi resistant models. To overcome this issue, we wondered whether the shared up-regulated RTK phenotype associated with BRAFi resistance could be exploited by using immune weapons as the antibody-dependent cell cytotoxicity (ADCC)-mediated effect of anti-RTKs antibodies, and kill tumor cells independently from the mechanistic roots. By using an in vitro model of BRAFi resistance, we detected increased membrane expression of EGFR, both at mRNA and protein level in 4 out of 9 BRAFi-resistant (VR) CM cultures as compared to their parental sensitive cells. Increased EGFR phosphorylation and AKT activation were observed in the VR CM cultures. EGFR signaling appeared dispensable for maintaining resistance, since small molecule-, antibody- and CRISPR-targeting of EGFR did not restore sensitivity of VR cells to BRAFi. Importantly, immune-targeting of EGFR by the anti-EGFR antibody cetuximab efficiently and specifically killed EGFR-expressing VR CM cells, both in vitro and in humanized mouse models in vivo, triggering ADCC by healthy donors' and patients' peripheral blood cells. Our data demonstrate the efficacy of immune targeting of RTKs expressed by CM relapsing on BRAFi, providing the proof-of-concept supporting the assessment of anti-RTK antibodies in combination therapies in this setting. This strategy might be expected to concomitantly trigger the crosstalk of adaptive immune response leading to a complementing T cell immune rejection of tumors.

Identification of CD160-TM as a tumor target on triple negative breast cancers: possible therapeutic applications

BackgroundDespite major therapeutic advances, triple-negative breast cancer (TNBC) still presents a worth prognosis than hormone receptors-positive breast cancers. One major issue relies in the molecular and mutational heterogeneity of TNBC subtypes that is reinforced by the absence of reliable tumor-antigen that could serve as a specific target to further promote efficient tumor cell recognition and depletion. CD160 is a receptor mainly expressed by NK lymphocytes and presenting two isoforms, namely the GPI-anchored form (CD160-GPI) and the transmembrane isoform (CD160-TM). While CD160-GPI is constitutively expressed on resting cells and involved in the generation of NK cells' cytotoxic activity, CD160-TM is neo-synthesized upon activation and promotes the amplification of NK cells' killing ability.MethodsCD160 expression was assessed by immunohistochemistry (IHC) and flow cytometry on TNBC patient biopsies or cell lines, respectively. Antibody (Ab)-mediated tumor depletion was tested in vitro by performing antibody-dependent cell cytotoxicity (ADCC) and phagocytosis (ADCP) assays, and in vivo on a TNBC mouse model.ResultsPreliminary data obtained by IHC on TNBC patients' tumor biopsies revealed an unconventional expression of CD160 by TNBC tumor cells. By using a specific but conformation-dependent anti-CD160-TM Ab, we established that CD160-TM, but not CD160-GPI, was expressed by TNBC tumor cells. A conformation-independent anti-CD160-TM mAb (22B12; muIgG2a isotype) was generated and selected according to pre-defined specificity and functional criterions. In vitro functional assays demonstrated that ADCC and ADCP could be induced in the presence of 22B12, resulting in TNBC cell line apoptosis. The ability of 22B12 to exert an in vivo anti-tumor activity was also demonstrated on a TNBC murine model.ConclusionsOur data identify CD160-TM as a tumor marker for TNBC and provide a rational for the use of anti-CD160-TM antibodies as therapeutic tools in this tumor context.

A phase Ib/II study of ASKB589 (anti-Claudin 18.2 [CLDN18.2] monoclonal antibody) combined with CAPOX and PD-1 inhibitor as first-line treatment for locally advanced, relapsed and metastatic gastric/gastro-esophageal junction (G/GEJ) adenocarcinoma.

317 Background: ASKB589 is a humanized IgG1 monoclonal antibody against Claudin 18.2 (CLDN18.2) with high affinity and enhanced antibody-dependent cytotoxicity. We report preliminary safety and efficacy data from an ongoing Phase Ib/II, dose-escalation and expansion study of ASKB589 combined with capecitabine, oxaliplatin(CAPOX) and Sintilimab as first-line treatment of G/GEJ adenocarcinoma (NCT05632939). Methods: The study enrolled G/GEJ adenocarcinoma patients(pts) with CLDN18.2 positive expression. The dose-escalation phase used a 3 + 3 design to determine the maximum tolerated dose (MTD). In expansion, the dose of ASKB589 selected for expansion was based on the safety, tolerability, pharmacokinetics and antitumor activity during escalation phase. Responses were assessed by RECIST 1.1 every 2 cycles (6 weeks). Adverse events (AEs) were graded using CTCAE v5.0. In escalation, pts received ASKB589 intravenously (IV) at doses of 6 mg/kg (n = 3) and 10 mg/kg (n = 6) every 3 weeks (Q3W) combined with CAPOX and Sintilimab. In expansion, all pts received ASKB589 IV at a dose of 6 mg/kg. Results: As of July 20, 2023, 9 pts were enrolled in escalation. No DLT was observed and thus the MTD was not identified. 9(100%)pts had treatment-related adverse events (TRAEs), the most common being nausea (77.8%), hypoproteinemia (66.7%), and hyponatremia (55.6%). While the majority of TRAEs were grade 1 or 2, 1 pt (11.1%) had a grade 3 TRAE (decreased neutrophils [10mg/kg]). In expansion, 26 pts with CLDN18.2 moderate-to-high expression (≥2+ membrane staining intensity in ≥40% of tumor cells) were included in the safety set. 24(92.3%) pts had TRAEs, the most common being nausea (65.4%), vomiting (53.8%), hypoproteinemia (53.8%), anemia (50.0%), fatigue (26.9%) and decreased neutrophils (23.1%). 3 pts (11.5%) had TRAEs of grade ≥3 including hypocalcemia, hypokalemia, decreased neutrophils, fatigue and bone marrow suppression. For the 15 evaluable pts who had at least one post-baseline tumor assessment, 12 pts (80.0%) achieved PR for an unconfirmed objective response rate (ORR) of 80.0%. 3 pts (20.0%) had SD for a disease control rate (DCR) of 100.0%. The PK of ASKB589 at doses of 6mg/kg and 10mg/kg combined with CAPOX and anti-PD1 therapy was consistent with that of monotherapy. Conclusions: ASKB589 combined with CAPOX and PD-1 inhibitor has manageable safety and promising antitumor activity. 6mg/kg is chosen as the recommended dose in subsequent studies. Clinical trial information: NCT05632939 .

A Review of Anti-CD20 Antibodies in the Management of B-Cell Lymphomas

Anti-CD20 monoclonal antibodies (mAbs) have revolutionized the treatment of lymphomas by improving the survival of patients, particularly in conjunction with chemotherapy. Until recently, the gold standard was based on the utilization of Rituximab (RTX) combined with chemotherapy. With our better understanding of monoclonal antibody (mAb) engineering, anti-CD20 mAb therapy has evolved to enhance clinical outcomes by improving pharmacokinetics, safety, activity and immunogenicity. Efforts to improve the on-targeting CD20 expressed on lymphomas through novel bioengineering techniques have led to the development of newer anti-CD20 mAbs that have accentuated complement-dependent cytotoxicity (CDC), antibody-dependent cell medicated cytotoxicity (ADCC), and/or a direct killing effect. There are several anti-CD20 monoclonal antibodies that have been evaluated for the treatment of lymphomas, some of which are now approved in addition to RTX.

Current and Future States of Natural Killer Cell-Based Immunotherapy in Hepatocellular Carcinoma.

Natural killer (NK) cells are innate lymphoid cells that exhibit high levels of cytotoxicity against NK-specific targets. NK cells also produce various cytokines, and interact with T cells, B cells, and dendritic cells to effectively serve as frontliners of the innate immune system. Produce various cytokines, and interact with T cells, B cells, and dendritic cells to effectively serve as frontliners of the innate immune system. Moreover, NK cells constitute the second most common immune cell in the liver. These properties have drawn significant attention towards leveraging NK cells in treating liver cancer, especially hepatocellular carcinoma (HCC), which accounts for 75% of all primary liver cancer and is the fourth leading cause of cancer-related death worldwide. Notable anti-cancer functions of NK cells against HCC include activating antibody-dependent cell cytotoxicity (ADCC), facilitating Gasdermin E-mediated pyroptosis of HCC cells, and initiating an antitumor response via the cGAS-STING signaling pathway. In this review, we describe how these mechanisms work in the context of HCC. We will then discuss the existing preclinical and clinical studies that leverage NK cell activity to create single and combined immunotherapies.

Zolbetuximab for Claudin18.2-positive gastric or gastroesophageal junction cancer.

Claudins (CLDNs) are a family of major membrane proteins that form components of tight junctions. In normal tissues, CLDNs seal the intercellular space in the epithelial sheets to regulate tissue permeability, paracellular transport, and signal transduction. Claudin18.2 (CLDN18.2), a member of the CLDN family, is expressed specifically in gastric mucosal cells in normal tissue, and its expression is often retained in gastric cancer cells. CLDN18.2 is ectopically expressed in many cancers other than gastric cancer such as esophageal cancer, pancreatic cancer, biliary tract cancer, non-small-cell lung cancer, and ovarian cancer. Structurally, CLDN18.2 is localized on the apical side of the cell membrane and has extracellular loops capable of binding monoclonal antibodies. Upon malignant transformation, CLDN18.2 is exposed to the cell surface of the whole membrane, which enables the binding of monoclonal antibodies. Based on these characteristics, CLDN18.2 was considered to be optimal for target therapy, and zolbetuximab was developed which is a first-in-class chimeric immunoglobulin G1 monoclonal antibody highly specific for CLDN18.2. It binds to CLDN18.2 on the tumor cell surface and stimulates cellular and soluble immune effectors that activate antibody-dependent cytotoxicity and complement-dependent cytotoxicity. Recently, zolbetuximab combined with chemotherapy demonstrated a survival benefit in patients with CLDN18.2-positive and HER-2-negative gastric or gastroesophageal junction cancers in the global phase III SPOTLIGHT and GLOW trials. From these clinically meaningful results, CLDN18.2-targeting therapy including zolbetuximab has attracted a lot of attention. In this review, we summarize the clinical implications of CLDN18.2-positive gastric or GEJ cancer, and CLDN18.2-targeting therapy, mainly for zolbetuximab.

Ovarian cancer treatment and natural killer cell-based immunotherapy.

Ovarian cancer (OC) is one of the malignant tumors that poses a serious threat to women's health. Natural killer (NK) cells are an integral part of the immune system and have the ability to kill tumor cells directly or participate indirectly in the anti-tumor immune response. In recent years, NK cell-based immunotherapy for OC has shown remarkable potential. However, its mechanisms and effects remain unclear when compared to standard treatment. To explore the value of NK cell-based immunotherapy in the treatment of OC, we conducted a literature review. In comparison to standard treatment, our focus was primarily on the current anti-tumor mechanisms, the clinical effect of NK cells against OC, factors affecting the structure and function of NK cells, and strategies to enhance the effectiveness of NK cells. We found that NK cells exert their therapeutic effects in OC through mechanisms such as antibody-dependent cell cytotoxicity, perforin release, and granule enzyme secretion. They also secrete IFN-γ and TNF-α or engage in Fas/FasL and TRAIL/TRAILR pathways, mediating the death of OC cells. In clinical trials, the majority of patients experienced disease stability with mild side effects after receiving NK cell-based immunotherapy, but there is still a lack of high-quality research evidence regarding its clinical effectiveness. OC and prior experience with standard treatments have an effect on NK cells, and it may be considered to maximize NK cell effects through the modulation of the tumor microenvironment or combination with other therapies. In this review, we have summarized the current evidence of NK cell applications in the treatment of OC. Furthermore, factors and strategies that influence and enhance the role of NK cell immunotherapy are discussed.

Composition and Dynamic Evolution of the Bone Marrow Microenvironment in AL Amyloidosis during Daratumumab Based Therapy

Background: Immunoglobulin light chain (AL) amyloidosis is caused by clonal B or plasma cells proliferating in the bone marrow that produce immunoglobulin (MIg) proteins that misfold and aggregate in tissue(s), leading to multi-organ failure. The monoclonal antibody targeting CD38, daratumumab is an approved and very efficient treatment in multiple myeloma and AL Amyloidosis by inducing plasma cell death through immune mediated mechanisms including complement and antibody-dependent cytotoxicity, direct apoptosis and T cell subsets modifications. While the role of the bone marrow microenvironment (BME) as a major oncogenic actor in multiple myeloma (MM) is well established, the BME composition and role in AL Amyloidosis is largely undefined. Here, we used single-cell RNA-seq combined with CITE-seq and TCR sequencing to characterize the immune ecosystem and microenvironment in AL amyloidosis in comparison to healthy donors, as well as in comparison to other plasma cell disorders including monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM) and multiple myeloma (MM). Methods: BM aspirates from 6 AL amyloidosis patients were collected at diagnosis or before treatment with daratumumab based therapy and 12 months post-treatment, alongside samples from 13 healthy donors, 5 MGUS, 9 SMM and 8 MM patients at diagnosis. We performed unbiased mRNA profiling (5' sequencing) and feature barcoding for cell surface proteins (CITE-seq) of bone marrow mononuclear cells using 10x Genomics. We processed data via the Cell Ranger pipeline, which grouped T cells into clonotypes based on shared TCR α/β sequences. Further data processing was done with Seurat, scRepertoire and Immunarch. Overall, 83703 cells were sequenced from 45 samples, including 3065 plasma cells and 80639 BME cells. Results: Compared to other plasma cell disorders (PCD) at diagnosis, AL BME had more cellular diversity and its plasma cells were more similar to normal plasma cells. They also had more TCR diversity than other PCD samples. When compared to healthy samples, AL BME had a trend towards more CD8+ T cells (p value = 0.078) at diagnosis, which became more pronounced 12 months post-treatment (p value = 0.012), and a trend towards fewer monocytes (p value = 0.036). When compared to PCD samples, AL BME tended to have more hematopoietic stem cells (p value = 0.018 vs MGUS, 0.052 vs SMM and 0.02 vs MM) and fewer NK cells (p values respectively 0.03, 0.059, 0.05) and CD4+ T cells (p values respectively 0.05, 0.018, 0.02) (Figure 1). The number of memory T cells and exhausted T cells were similar. Importantly, AL samples were significantly enriched in early/young CD4 memory T cells (p value < 2.6e-16) and NK/T like CD8+ T cells (p value = 7.99e-11) suggesting an ongoing and active T cell mediated immune response. We next analyzed serial samples from 4 AL patients at diagnosis and ~12 months after Daratumumab-based therapy, with half the patients achieving complete hematologic and organ responses (CR) and half with incomplete responses. We found broad changes in the BME, but in particular, there were more CD4+ and CD8+ T cells at 12 months expressing cytokine signaling genes (p value 1.19e-35) and adaptive immune system pathway genes (p value = 2.59e-33). Importantly, the distributions of CD8+ cells and T cell clonotypes were different between patients achieving CR and those who did not, underlying the role of the immune microenvironment and especially CD8+ T cells in the pathogenesis of the disease and response to Daratumumab based treatment. Conclusion: We identified unique characteristics of the BME in AL amyloidosis and dynamic changes within the T cell compartment following Daratumumab based therapy. Further validations are needed to evaluate the potential of BME profiling to diagnose the disease, predict treatment response and potentially develop new therapeutic strategies in AL Amyloidosis.

Expansion, Persistence, and Characteristics of Autologous, Bhv-1100 Armored Memory-like NK Cells Infused Prior to Autologous Stem Cell Transplant in MRD+ Multiple Myeloma Patients: A First-in-Human Trial

Introduction/Background Autologous stem cell transplant (ASCT) improves MRD negativity and prolongs progression-free survival in patients with multiple myeloma (MM) in their first or second remission following induction chemotherapy. MM NK cells are dysfunctional, negatively impacting outcomes. BHV-1100, a novel Antibody Recruiting Molecule ( ARM), binds to CD38 and recruits NK cells for antibody-dependent cell cytotoxicity (ADCC) without inducing fratricide. Allogeneic, cytokine induced memory-like (CIML) NK cells effectively treat myeloid disorders, however, it is not known if autologous CIML NK cells, when coated with BHV-1100, would further improve ASCT outcomes in MM. Methods We designed a first-in-human study of autologous CIML NK cells coated ex-vivo with BHV-1100 for MRD+, MM patients undergoing ASCT following first or second remission. NK cells were isolated from non-mobilized leukapheresis on day -1 (prior to melphalan for HCT) using CD3 depletion followed by CD56 positive selection with Miltenyi's CliniMACS. The NK cells were incubated overnight (12-16 hours) with IL-12 (10ng/ml), IL-15 (100ng/ml), and IL-18 (50ng/ml) to induce CIML differentiation, washed and subsequently coated with BHV-1100 for one hour prior to infusion. The product was infused fresh on D0 after standard melphalan 200 mg/m2 myeloablative conditioning and followed by stem cell infusion. Low dose IL2 (1 mIU/m 2) was administered SQ starting on D+1, QOD for a total of 7 doses. Results This is an ongoing trial (NCT04634435) with a median follow-up of 191 days. We are herein reporting data on the in vivo expansion and functional characterization of ARMored CIML NK for the first 5 patients. CIML NK cells were manufactured with a 100% success rate and infused at a target dose of 5-10x10 6 cells/kg-body weight, 24 hours after 200 mg/m 2 melphalan administration. Patients received between 3.9-6.0x10 6/Kg stem cells. Engraftment based on recovery of neutrophil count occurred on D+12-D+14. There was a 3-fold expansion of NK cells in the peripheral blood from D+7 (from 12% to 42%) to D+28 that persisted until D+60 (25% total PBMC, Fig. 1A). Most expanded NK cells were CD56 dim, CD16 high, KIR high and CD57 high. CD57 and KIR expression increased over time from D+7 to D+60, whereas NKG2A expression decreased, indicating the expansion of mature, activated, and cytotoxic NK cells. Regulatory T cells increased by D+7 (3% vs 15% total PBMC) and returned to baseline after D+14 most likely reflecting the effect of IL-2 treatment. The functional capacity of the infused product was tested in vitro against MOLP8 MM cell line. The BHV-1100 ARMored CIML NK cells showed increased IFN (53% vs 48% at 0H and 53% vs 44% at 24H) and CD107a (Fig.1B) (26% vs 13% at 0H and 34% vs 15% at 24H) production compared to untreated CIML NK cells and the product was stable for 24 hours. Conclusion Autologous, BHV-1100 ARMored CIML NK cells have enhanced anti-MM activity as well as expand and persist in vivo peaking at D+28 after infusion. This represents an innovative approach to boost autologous cancer immunosurveillance in the context of ASCT. Aside from anticipated infusion reactions, no severe/unexpected adverse events were noted; longer follow-up is required to assess safety and efficacy.

Meta-Analysis of Randomized Controlled Trials to Evaluate the Incidence of Pneumonia, Upper Respiratory Tract Infections and Febrile Neutropenia in Patients with Multiple Myeloma Treated with Isatuximab

Introduction Multiple myeloma is a cancer of plasma cell and accounts for approximately 13% of all haematologic malignancies. Isatuximab is an anti CD38 IgG1 monoclonal antibody which binds to and kills myeloma cells through antibody-dependent cellular-mediated cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and direct induction of apoptosis. The incorporation of isatuximab to standard treatment in patients with relapsed refractory multiple myeloma (RRMM) has shown to significantly improve efficacy, with notable toxicities. We performed a combined analysis of randomized controlled trials (RCT) to determine the risk of pneumonia and upper respiratory tract infections (URTIs) and febrile neutropenia in patients with RRMM treated with isatuximab. Methods We systematically conducted a comprehensive literature search using MEDLINE, EMBASE databases and meeting abstracts from inception through June 30 th, 2023. Phase III RCTs utilizing isatuximab in patients with relapsed or refractory multiple myeloma that mention pneumonia, URTIs and febrile neutropenia as adverse effects were incorporated in the analysis. Mantel-Haenszel (MH) method was used to calculate the estimated pooled risk ratio (RR) with 95% confidence interval (CI). Heterogeneity was assessed with Cochran's Q- statistic. Random effects model was applied. Results A total of 600 patients with RRMM from 2 phase III RCTs (ICARIA-MM and IKEMA) were eligible. ICARIA-MM and IKEMA compared isatuximab (I) + pomalidomide (P) + dexamethasone (d) vs Pd, and isatuximab (I) + Carfilzomib (K) + dexamethasone (d) vs Kd respectively. The randomization ratios were 1:1 in ICARIA-MM, and 3:2 in IKEMA studies. The I 2 statistic for heterogeneity was 0, suggesting homogeneity among RCT. The incidence of any-grade pneumonia was 30.1% in study group vs 25.8% in control group (RR, 1.19; 95% CI: 0.91 - 1.54; P=0.20). High-grade pneumonia rate was nearly 4% higher in isatuximab group compared to control arm but RR was not statistically significant at 1.26 (95% CI: 0.90 - 1.75; P = 0.18). More events of any-grade URTIs were reported in study arm (36.8%) versus 23.2% in control arm with RR of 1.55 (95% CI: 1.20- 2.01; P = 0.0008). The incidence of high-grade URTIs was low in both groups: 3.3% in study group versus 1.47% in control arm (RR, 2.25; 95% CI: 0.72 - 6.98; P = 0.16). Febrile neutropenia was noted in 20 (6%) of patients in study group compared to 5 (1.8%) in control group with RR of 3.52 (95% CI: 1.40- 8.83; P = 0.007). Conclusions Our meta-analysis showed that the addition of isatuximab to standard anti-myeloma regimen contributed to higher incidence of any-grade URTIs and febrile neutropenia in patients with RRMM, with RR of 1.55 for any-grade URTIs and RR of 3.5 for febrile neutropenia. However, risks of high-grade URTIs, any-grade and high-grade pneumonia were found to be statistically not significant between the two groups. Early recognition and proper supportive care are required to improve any-grade URTIs and febrile neutropenia.