Antibody-dependent Cellular Cytotoxicity Activity Research Articles

Identification of an Immunodominant B-Cell Epitope in African Swine Fever Virus p30 Protein and Evidence of p30 Antibody-Mediated Antibody Dependent Cellular Cytotoxicity

Identification of an Immunodominant B-Cell Epitope in African Swine Fever Virus p30 Protein and Evidence of p30 Antibody-Mediated Antibody Dependent Cellular Cytotoxicity

Abstract LB130: Identification of fully human CCR8 antibodies that demonstrate excellent ADCC function, potent blocking activity, and robust anti-tumor immune responses

Abstract Immune checkpoint inhibitors have demonstrated significant benefits in treating various types of tumors. However, a considerable number of patients do not respond to this therapy. One proposed mechanism for resistance to immune checkpoint inhibitors is the infiltration of large numbers of regulatory T cells (Tregs) into tumors, which hinders the development of effective antitumor immunity and is often linked to poor prognosis. CCR8 is a G-protein-coupled receptor (GPCR) which is predominantly expressed on Tregs infiltrating tumors. The development of CCR8-specific human antibodies to deplete CCR8+ Tregs is a promising therapeutic strategy for cancer treatment. Here, we report the development and characterization of fully human monoclonal antibodies targeting CCR8 that exhibit potent antibody-dependent cellular cytotoxicity (ADCC) activity. These antibodies were generated in CCR8 knock-out RenMab® mice, which were immunized with native CCR8 antigen prepared via a proprietary cell-free membrane protein synthesis technology. Epitope mapping of the antibodies revealed recognition of a unique epitope on the N-terminus extracellular domain of CCR8. Binding assays indicated cross-reactivity of the antibodies across multiple species; importantly, however, these antibodies did not recognize related chemokine receptors, such as CCR2, CCR4, CCR5, or CX3CR1. In vitro, CCR8 antibodies effectively inhibited calcium flux induced by CCL1 binding to the CCR8 receptor. Furthermore, introduction of DE (S239D, I332E) mutations in the Fc region enhanced ADCC activity of these antibodies. To compare the efficacy of these antibodies with anti-CCR8 benchmarks in vivo, we generated benchmark analogs in-house with the same DE mutations and established syngeneic MC38 tumor models in humanized B-hCCR8 mice. In this assay, our CCR8 antibodies demonstrated potent anti-tumor activity. Notably, one mutant clone with optimized developability demonstrated robust inhibition of tumor growth when compared to both benchmark analogs and its original clone. Taken together, our fully human CCR8 antibodies demonstrate enhanced ADCC function, strong blocking activity, and superior in vivo anti-tumor activity, which may support further development into a novel cancer immunotherapy targeting Tregs. Citation Format: Huichao Liang, Yanan Guo, Meimei Yin, Aki Kawagishi, Takaho Terada, Yoshihiko Kihara, Toru Kanke, Chaoshe Guo, W. Frank An, Yi Yang. Identification of fully human CCR8 antibodies that demonstrate excellent ADCC function, potent blocking activity, and robust anti-tumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB130.

Abstract CT162: Trial in progress: A phase I, open-label, multicenter study of ZL-1218, an anti-CCR8 IgG1, as a single agent and as combination therapy with anti-PD-1 antibody to evaluate the safety, tolerability, and pharmacokinetics in subjects with advanced solid tumor

Abstract Background ZL-1218 is an anti-chemokine receptor 8 (CCR8) humanized IgG1 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) activity. CCR8 demonstrates a preferential expression in tumor-associated Tregs over peripheral Tregs. Thus, targeting CCR8 may allow for specific depletion of intratumoral Tregs without impacting peripheral Tregs/other immune cells. ZL-1218 is intended to decrease the intratumoral Tregs by binding to both high- and low-expressing CCR8+ cells and eliciting potent ADCC activity. ZL-1218 blocked CCR8 ligand CCL1 from binding to CCR8, and reduced Treg recruitment by blocking chemotaxis of CCR8+ cells (unpublished data). Additionally, ZL-1218 demonstrated enhanced antitumor activity in preclinical models in combination with anti-PD-1. Methods This is a Phase I study of ZL-1218 as a single agent and in combination with pembrolizumab (P) to evaluate the safety, tolerability, pharmacokinetics/pharmacodynamics, and preliminary antitumor activity in subjects with advanced solid tumor malignancies. The study consists of Part 1 Dose Escalation and Part 2 Cohort Expansion. In Part 1, subjects will be treated with escalating doses of ZL-1218 alone and in combination with a fixed dose of P to identify the maximum tolerated dose (MTD) and/or the recommended expansion dose (RD). The Bayesian optimal interval (BOIN) design is used and adopted with overdose control. Upon progression in the single agent group, a subject may enroll into combination therapy of ZL-1218 with P. Subjects will start at their respective single agent dose or the cleared combination dose level, whichever is lower. Part 2 will explore the combination with pembrolizumab in subjects with selected tumor types, with or without prior CPI therapy. Major inclusion criteria include: (1) Subjects must have histologically confirmed and documented diagnosis of locally advanced unresectable/metastatic advanced solid tumor that is refractory/intolerant to standard treatment, or for which no standard treatment exists, and no greater than 3 prior lines of therapy in the relapse/refractory setting (2). Subjects must have at least one target lesion as defined by RECIST v1.1 on CT, PET/CT, or MRI scan. (3) Subjects must have a site of disease which is not previously irradiated and is safe and amenable to biopsy. Major exclusion criteria include: (1) Prior exposure to CCR8 inhibitor, or severe hypersensitivity (≥ Grade 3) to P and/or any of its excipients. (2) Subjects with symptomatic or uncontrolled brain metastasis requiring concurrent treatment, impaired major organ functions, active autoimmune disease, or active infections. Part 1 Dose Escalation is ongoing in the US and EU. Citation Format: Elena Garralda, Valentina Boni, David Vincente Baz, Arvind Chaudhry, Ignacio Gil Bazo, Martin Gutierrez, Jyoti Malhotra, Casal Guzman Alonso, Valentina Gambardella, Michael Chisamore, Renke Zhou, Maria Tea, Deborah Doroshow. Trial in progress: A phase I, open-label, multicenter study of ZL-1218, an anti-CCR8 IgG1, as a single agent and as combination therapy with anti-PD-1 antibody to evaluate the safety, tolerability, and pharmacokinetics in subjects with advanced solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT162.

Abstract LB129: Preclinical development of a bispecific antibody-trap selectively targeting CD38 and CD47 for treating hematologic malignancies

Abstract CD38 and CD47 are co-expressed in various hematologic malignancies including multiple myeloma (MM), B-cell non-Hodgkin lymphoma (NHL), B-cell acute lymphoblastic leukemia (ALL), T-cell ALL and B-cell chronic lymphocytic leukemia (CLL). We present, herein, several CD38 antibody - CD47 ligand trap bispecific antibodies (BsAbs) in an ADCC-enhanced IgG1 form that harbor high CD38 binding affinity, low CD47 binding affinity, along with negligible affinity to red blood cells (RBCs) and no induction of RBCs agglutination. Using flow cytometry and Biolayer interferometry (BLI), we confirmed that our BsAbs can simultaneously bind to both CD38 and CD47, and that binding to either antigen first does not prevent binding to the other antigen subsequently. On CD47+/CD38low Jurkat (T leukemic) cells, our BsAbs reveal less blocking effect on the CD47/SIRPα interaction than a monospecific CD47 ligand trap molecule based on which our BsAbs were designed; conversely, on CD38/CD47 double-positive Reh (pro-B ALL) cells, our BsAbs reveal 200-500-fold greater blocking effect on the CD47/SIRPα interaction than the same monospecific CD47 ligand trap molecule, indicating that the inhibition of CD47/SIRPα by our BsAbs is depended on the engagement of CD38 on tumor cells. On CD47+/CD38+ Daudi (B NHL) lymphoma cells and L-1236 (B Hodgkin) lymphoma cells, four of our BsAbs inhibited CD38 enzymatic activity, which is responsible for immune-suppressive adenosine production in tumor microenvironment. Our BsAbs presented potent dose-dependent antibody-dependent cellular cytotoxicity (ADCC) against CD47+/CD38+ Raji (B NHL) and NCI-H929 (B plasmacytoma myeloma) cells, while revealing negligible ADCC activity against CD47+/CD38− cells, indicating low off-tumor toxicity. Likewise, our BsAbs also harbor potent antibody-dependent cellular phagocytosis (ADCP) activity against Raji NHL cells, varied degree of complement-dependent cytotoxicity (CDC) activity and direct apoptosis induction against Daudi NHL cells. In a mouse xenograft model harboring NCI-H929 multiple myeloma, our BsAbs produced more potent tumor growth inhibition, than monospecific anti-CD47 ligand trap, anti-CD38 (isatuximab) or combination of isatuximab and anti-CD47 ligand trap, at the same molecular dose per body weight. Notably, clone IMM5605-12C10 eradicated all tumors and achieved 100% complete response rate in a group of six mice. In summary, simultaneous targeting CD38 and CD47 using our CD38 antibody - CD47 ligand trap bispecific design in an ADCC-enhanced IgG1 form presents a novel multimodal approach for treating hematologic malignancies that are resistant to anti-CD38 antibodies. Citation Format: Wenzhi Tian, Song Li, Dianze Chen, Yanan Yang, Huiqin Guo, Dandan Liu, Ruliang Zhang, Fan Zhang. Preclinical development of a bispecific antibody-trap selectively targeting CD38 and CD47 for treating hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB129.

Abstract 2668: CBO421: A novel drug Fc-conjugate to prevent tumor immune evasion via the CD73/adenosine pathway

Abstract Background CD73 (5’-nucleotidase or NT5E) is a cell surface enzyme responsible for the rate limiting step in adenosine production; the conversion of adenosine monophosphate (AMP) into adenosine. Adenosine contributes to immune evasion by solid tumors by concentrating immunosuppressive adenosine in the tumor microenvironment. CBO421, a Drug Fc-Conjugate (DFC), comprises a novel small molecule CD73 inhibitor stably conjugated to an immune-silent human IgG1 Fc, combining the strengths of small molecule inhibitors and CD73-targeting monoclonal antibodies (mAbs) for potential best-in-class efficacy. Methods CBO421’s binding affinity to human CD73 and Fc receptors was assessed by surface plasmon resonance. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated using a commercial kit. Immunophenotyping and binding to cancer cells was measured by flow cytometry. Inhibition of soluble and membrane-bound CD73 on human PBMCs and mouse cancer cells was determined by measuring free phosphate levels. Functional activity was measured using PBMC rescue assays. In vivo efficacy of CBO421 monotherapy was evaluated in a syngeneic mouse model. Results CBO421 exhibited a binding affinity to human CD73 (KD = 0.8 nM) that was comparable to or greater to the affinity observed with small molecule inhibitors (AB680, OP-5244) and CD73-targeting mAbs (oleclumab, mupadolimab biosimilars). As expected, human FcRn binding of CBO421 was comparable to the unconjugated Fc and the full-length wild-type IgG1 control mAb. CBO421 binding to human Fc gamma receptors and ADCC activity was significantly reduced or abolished compared to the control mAb, such that it poses a minimal risk for undesired autoimmune activity. CBO421 potently inhibited soluble CD73 and CD73 expressed on human MDA-MB-231 and mouse EMT6 cancer cells. Immunophenotyping revealed that CD73 expression was primarily observed on CD8+ T and B cells from human PBMCs. In functional PBMC rescue assays, CBO421 demonstrated potent reactivation of AMP- or ATP-suppressed CD4+ or CD8+ T cells at levels comparable to small molecule inhibitors and greater than anti-CD73 mAb comparators. CBO421 monotherapy demonstrated a dose-dependent reduction in tumor volume in a syngeneic mouse model using MC38, a colorectal cancer cell line. Conclusions CBO421 exhibited strong binding and potent inhibition of both soluble and membrane-bound CD73, differentiating it from mAb CD73 inhibitors. The proprietary immune-silent Fc of CBO421 did not affect FcRn binding but substantially reduced Fc gamma receptor binding, leading to the elimination of ADCC activity to prevent undesired immune cell depletion in host tissues. CBO421 demonstrated high potency in functional cell-based assays and robust antitumor efficacy in a syngeneic mouse model. Currently, CBO421 is being advanced as a clinical development candidate for the treatment of solid cancers. Citation Format: Amanda Almaguer, Doug Zuill, Simon Döhrmann, James Levin, Nicholas Dedeic, Elizabeth Abelovski, Joanne Fortier, Qiping Zhao, Maria Hernandez, Karin Amundson, Madison Moniz, Hongyuan Chen, Dhanya Panickar, Thanh Lam, Tom Brady, Allen Borchardt, Grayson Hough, Jeffrey B. Locke, Jason N. Cole, Leslie W. Tari. CBO421: A novel drug Fc-conjugate to prevent tumor immune evasion via the CD73/adenosine pathway [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 2668.

Abstract 6582: Preclinical characterization of IPH45, a novel topoisomerase I inhibitor ADC targeting Nectin-4 for the treatment of Nectin-4 expressing tumors

Abstract Background: Nectin-4 is a cell membrane adhesion protein overexpressed in several solid tumors, including urothelial, breast, lung, ovarian, and pancreatic cancers, with limited expression in normal tissues, making it an ideal target for antibody-drug conjugates (ADC). Enfortumab Vedotin (EV, PADCEV®) is an ADC targeting Nectin-4 with a monomethyl auristatin E (MMAE) payload approved for the treatment of patients with urothelial carcinoma (UC), but with limited clinical activity reported beyond UC. In addition, EV causes peripheral neuropathy and skin toxicity, leading to treatment discontinuation. To overcome these limitations and the mechanism of resistance to EV, IPH45, a novel topoisomerase I inhibitor ADC targeting Nectin-4 with an improved therapeutic index, was developed. Methods: In vitro studies were performed to investigate internalization, tumor cell killing and bystander effect, antibody-dependent cellular cytotoxicity (ADCC) and complement activation. In vivo efficacy studies were performed on cell line-derived xenografts (CDX) of triple negative breast cancer, patient-derived xenografts (PDX) of urothelial carcinoma and syngeneic tumor models expressing human Nectin-4. IPH45 efficacy was compared to EV. Toxicology studies to determine the dose range were conducted in rat and non-human primates (NHP). Results: IPH45 consists of a humanized IgG1 targeting a unique epitope in human Nectin-4, non-overlapping with EV, a cleavable linker, and a topoisomerase I inhibitor with a high drug-to-antibody ratio (DAR). In vitro, IPH45 is internalized upon binding to its target and induces direct killing of Nectin-4 expressing tumor cells as well as bystander killing of Nectin-4 non-expressing tumor cells in mixed culture. IPH45 mediates ADCC and complement-mediated killing. IPH45 demonstrates activity in CDX and PDX models with similar effective dose as EV. Moreover, IPH45 shows anti-tumor efficacy in vitro and in vivo in primary and secondary EV resistant models. In dose-range finding toxicology studies in rats and NHP, IPH45, unlike EV, does not elicit skin toxicity. IPH45 remains well tolerated up to the highest dose tested, suggesting a potentially larger therapeutic index than MMAE-based ADCs. Pharmacokinetic analyses in mice, rats, and NHP show minimal release of free toxin in the serum and half-life compatible with Q2W or less frequent dosing schedules in clinic. Conclusion: IPH45 is a topoisomerase I ADC targeting Nectin-4 with the potential for larger therapeutic index than EV, improved safety and dosing regimen, and ability to overcome primary and secondary resistance to EV or other Nectin-4 ADC in development. IPH45 is progressing to the clinic where the translatability of this unique profile into improved patient outcomes will be assessed across indications. Citation Format: Romain Remark, Cécile Bonnafous, Laura Chiossone, Caroline Soulas, Cyril Perrier, Guillaume Habif, Sivan Bokobza, Aurélie Maguer, Rachel Courtois, Julie Lopez, Grégory Fenaux, Olivier Benac, Barbara Carrette, Robin Letay-Drouet, Raja Bonifay, Séverine Augier, Léa Simon, Benjamin Rossi, Ariane Morel, Agnès Represa, Nicola Beltraminelli, Yannis Morel, Carine Paturel, Eric Vivier. Preclinical characterization of IPH45, a novel topoisomerase I inhibitor ADC targeting Nectin-4 for the treatment of Nectin-4 expressing tumors [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 6582.

Abstract 1863: α-ABN501, a novel antibody targeting claudin-3, demonstrates the potential to be a new therapeutic option for small cell lung cancer

Abstract Small Cell Lung Cancer (SCLC) is the most aggressive and lethal type of lung cancer, characterized by a poor prognosis, with a 5-year survival rate of only 7% for overall SCLC patients. Limited treatment options such as chemotherapy and immune-checkpoint inhibitor therapy are available for SCLC patients, very often followed by rapid recurrence and metastasis. Thus, new effective treatment options for SCLC remain high unmet medical needs. Overexpression of claudin-3 (CLDN3), one of the tight junction proteins, is observed in various types of cancers. The abnormal conformation of tumor cell growth leads to the external exposure of CLDN3, in contrast, the normal cells that maintain high polarity do not expose CLDN3 externally. Therefore, CLDN3 could be a potential therapeutic target and biomarker. Based on the analysis of the expression pattern of CLDN3 using a single-cell RNA sequencing dataset derived from SCLC patients, we found that CLDN3 could be a superior and unique target of SCLC compared to others (e.g., DLL3 and B7H3). Hence, we developed a fully human monoclonal antibody, afucosylated ABN501 (α-ABN501), which has high specificity and strong affinity to CLDN3-expressing cells. The α-ABN501 antibody showed CLDN3-dependent killing effects against the SCLC cells, expressing different levels of CLDN3, as assessed via the antibody-dependent cellular cytotoxicity (ADCC) assay wherein using NK cells expressing CD16a (NK-92MI-CD16a), peripheral blood mononuclear cells (PBMC), and primary NK cells. Further, the efficacy study in the CLDN3-expressing SCLC cell derived xenograft mouse model revealed that α-ABN501 was highly efficacious in inhibition of tumor growth. Collectively, we elucidated that the ADCC activity of α-ABN501 was dependent on the expression level of CLDN3 and especially specific to the SCLC cells, which underlines significant suppression of tumor growth. Present study data sets suggest that the α-ABN501 can be a potential new therapeutic option for SCLC. In addition, the α-ABN501 antibody is currently under way of development into new drug platforms for further applications. Citation Format: Jae Choi, Sunghyun Hong, Ha Yeon Park, Jaemun Kim, Sangin Lee, Myeung-Ryun Seo, Sang-Beom Bang, Hyo-Jin Shin, Kwang-il Jeong, Hobin Yang, Sungyoul Hong, Jun Young Choi, Na Young Kim, Young Kee Shin, Saehyung Lee. α-ABN501, a novel antibody targeting claudin-3, demonstrates the potential to be a new therapeutic option for small cell lung cancer [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 1863.

Efbalropendekin Alfa enhances human natural killer cell cytotoxicity against tumor cell lines in vitro.

IL-15 has shown preclinical activity by enhancing the functional maturation of natural killer (NK) cells. Clinical evaluation of the potential anticancer activity of most cytokines, including IL-15, has been limited by low tolerability and rapid in vivo clearance. Efbalropendekin Alfa (XmAb24306) is a soluble IL15/IL15-receptor alpha heterodimer complex fused to a half-life extended Fc domain (IL15/IL15Rα-Fc), engineered with mutations to reduce IL-15 affinity for CD122. Reduced affinity drives lower potency, leading to prolonged pharmacodynamic response in cynomolgus monkeys. We show that in vitro, human NK cells treated with XmAb24306 demonstrate enhanced cytotoxicity against various tumor cell lines. XmAb24306-treated NK cells also exhibit enhanced killing of 3D colorectal cancer spheroids. Daratumumab (dara), a monoclonal antibody (mAb) that targets CD38 results in antibody-dependent cellular cytotoxicity (ADCC) of both multiple myeloma (MM) cells and NK cells. Addition of XmAb24306 increases dara-mediated NK cell ADCC against various MM cell lines in vitro. Because NK cells express CD38, XmAb24306 increases dara-mediated NK cell fratricide, but overall does not negatively impact the ADCC activity against a MM cell line likely due to increased NK cell activity of the surviving cells. These data show that XmAb24306 increases direct and ADCC-mediated human NK cell cytotoxicity in vitro.

SARS-CoV-2 infection triggers more potent antibody-dependent cellular cytotoxicity (ADCC) responses than mRNA-, vector-, and inactivated virus-based COVID-19 vaccines.

Neutralizing antibodies (NAbs) are elicited after infection and vaccination and have been well studied. However, their antibody-dependent cellular cytotoxicity (ADCC) functionality is still poorly characterized. Here, we investigated ADCC activity in convalescent sera from infected patients with wild-type (WT) severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) or omicron variant compared with three coronavirus disease 2019 (COVID-19) vaccine platforms and postvaccination breakthrough infection (BTI). We analyzed ADCC activity targeting SARS-CoV-2 spike (S) and nucleocapsid (N) proteins in convalescent sera following WT SARS-CoV-2-infection (n = 91), including symptomatic and asymptomatic infections, omicron-infection (n = 8), COVID-19 vaccination with messenger RNA- (mRNA)- (BNT162b2 or mRNA-1273, n = 77), adenovirus vector- (n = 41), and inactivated virus- (n = 46) based vaccines, as well as post-mRNA vaccination BTI caused by omicron (n = 28). Correlations between ADCC, binding, and NAb titers were reported. ADCC was elicited within the first month postinfection and -vaccination and remained detectable for ≥3 months. WT-infected symptomatic patients had higher S-specific ADCC levels than asymptomatic and vaccinated individuals. Also, no difference in N-specific ADCC activity was seen between symptomatic and asymptomatic patients, but the levels were higher than the inactivated vaccine. Notably, omicron infection showed reduced overall ADCC activity compared to WT SARS-CoV-2 infection. Although post-mRNA vaccination BTI elicited high levels of binding and NAbs, ADCC activity was significantly reduced. Also, there was no difference in ADCC levels across the four vaccines, although NAbs and binding antibody titers were significantly higher in mRNA-vaccinated individuals. All evaluated vaccine platforms are inferior in inducing ADCC compared to natural infection with WT SARS-CoV-2. The inactivated virus-based vaccine can induce N-specific ADCC activity, but its relevance to clinical outcomes requires further investigation. Our data suggest that ADCC could be used to estimate the extra-neutralization level against COVID-19 and provides evidence that vaccination should focus on other Fc-effector functions besides NAbs. Also, the decreased susceptibility of the omicron variant to ADCC offers valuable guidance for forthcoming efforts to identify the specific targets of antibodies facilitating ADCC.

Potent antibody-dependent cellular cytotoxicity of a V2-specific antibody is not sufficient for protection of macaques against SIV challenge.

Fc-mediated antibody effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), can contribute to the containment HIV-1 replication but whether such activities are sufficient for protection is unclear. We previously identified an antibody to the variable 2 (V2) apex of the HIV-1 Env trimer (PGT145) that potently directs the lysis of SIV-infected cells by NK cells but poorly neutralizes SIV infectivity. To determine if ADCC is sufficient for protection, separate groups of six rhesus macaques were treated with PGT145 or a control antibody (DEN3) by intravenous infusion followed five days later by intrarectal challenge with SIVmac239. Despite high concentrations of PGT145 and potent ADCC activity in plasma on the day of challenge, all animals became infected and viral loads did not differ between the PGT145- and DEN3-treated animals. To determine if PGT145 can protect against a neutralization-sensitive virus, two additional groups of six macaques were treated with PGT145 and DEN3 and challenged with an SIVmac239 variant with a single amino acid change in Env (K180S) that increases PGT145 binding and renders the virus susceptible to neutralization by this antibody. Although there was no difference in virus acquisition, peak and chronic phase viral loads were significantly lower and time to peak viremia was significantly delayed in the PGT145-treated animals compared to the DEN3-treated control animals. Env changes were also selected in the PGT145-treated animals that confer resistance to both neutralization and ADCC. These results show that ADCC is not sufficient for protection by this V2-specific antibody. However, protection may be achieved by increasing the affinity of antibody binding to Env above the threshold required for neutralization.

Development of therapeutic monoclonal antibodies against DKK1 peptide-HLA-A2 complex to treat human cancers

BackgroundTargeted immunotherapy with monoclonal antibodies (mAbs) is an effective and safe method for the treatment of malignancies. Development of mAbs with improved cytotoxicity, targeting new and known tumor-associated antigens, therefore...

Engineering of CD34+ progenitor-derived natural killer cells with higher-affinity CD16a for enhanced antibody-dependent cellular cytotoxicity

Background aimsNatural killer (NK) cell transfer is a promising cellular immunotherapy for cancer. Previously, we developed a robust method to generate large NK cell numbers from CD34+ hematopoietic stem and progenitor cells (HSPCs), which exhibit strong anti-tumor activity. However, since these cells express low levels of the Fc receptor CD16a in vitro, antibody-dependent cellular cytotoxicity (ADCC) by these cells is limited. To broaden clinical applicability of our HSPC-NK cells toward less NK-sensitive malignancies, we aimed to improve ADCC through CD16a transduction. MethodsUsing wildtype and S197P mutant greater-affinity (both with V158) CD16a retroviral transgenes (i.e., a cleavable and noncleavable CD16a upon stimulation), we generated CD16a HSPC-transduced NK cells, with CD34+ cells isolated from umbilical cord blood (UCB) or peripheral blood after G-CSF stem cell mobilization (MPB). CD16a expressing NK cells were enriched using flow cytometry–based cell sorting. Subsequently, phenotypic analyses and functional assays were performed to investigate natural cytotoxicity and ADCC activity. ResultsMean transduction efficiency was 34% for UCB-derived HSPCs and 20% for MPB-derived HSPCs, which was enriched by flow cytometry-based cell sorting to >90% for both conditions. Expression of the transgene remained stable during the entire NK expansion cell generation process. Proliferation and differentiation of HSPCs were not hampered by the transduction process, resulting in effectively differentiated CD56+ NK cells after 5 weeks. Activation of the HSPC-derived NK cells resulted in significant shedding of wildtype CD16a transcribed from the endogenous gene, but not of the noncleavable mutant CD16a protein expressed from the transduced construct. The mean increase of CD107+IFNγ+ expressing NK cells after inducing ADCC was tenfold in enriched noncleavable CD16a HSPC-NK cells. Killing capacity of CD16a-transduced NK cells was significantly improved after addition of a tumor-targeting antibody in tumor cell lines and primary B-cell leukemia and lymphoma cells compared to unmodified HSPC-NK cells. ConclusionsTogether, these data demonstrate that the applicability of adoptive NK cell immunotherapy may be broadened to less NK-sensitive malignancies by upregulation of CD16a expression in combination with the use of tumor-targeting monoclonal antibodies.

Daratumumab in the Treatment of Multiple Myeloma

A monoclonal antibody called Daratumumab (trade name: Darzalex) was created by Janssen Biotech, a division of Johnson & Johnson. The U.S. Food and Drug Administration originally approved it in November 2015 as a treatment for multiple myeloma in patients who had already tried at least three other lines of treatment. Daratumbabab has transformed multiple myeloma treatment since it was approved, and it has now acquired further approvals for a number of applications, reaffirming its place as a prominent therapeutic choice in the management of this challenging condition. It has been very effective in combating multiple myeloma. Although certain symptoms are present, its efficacy is still quite good. Daratumumab's ability to provide positive results in patients with newly diagnosed multiple myeloma who are ineligible for autologous stem-cell transplantation has been further supported by the Maia trial, a crucial study in this area. Daratumumab works by targeting CD38, a cell surface glycoprotein that is abundantly expressed in malignant plasma cells. This causes antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and direct activation of death. Daratumumab is becoming more and more important as a good therapeutic choice for people with multiple myeloma as a result of a thorough understanding of its effectiveness, symptoms, and mechanism of action.

Iberdomide Enhances Dara Mediated Cytotoxicity through Upregulation of CDC Activity and Elevated NK Cell Mediated ADCC

Background: Multiple Myeloma (MM) is an incurable hematological malignancy with current treatments comprising combination regimens, typically featuring immunomodulatory agents (IMiDs) due to their potent anti-tumor activity and immunomodulatory effects. Clinical studies utilizing the combination of IMiD agents and daratumumab (DARA), an anti-CD38 monoclonal antibody, have shown improved patient outcomes for MM. However, patients ultimately relapse demonstrating the need for novel therapies. Iberdomide (IBER) is a novel cereblon E3 ligase modulator. It promotes faster and deeper degradation of the substrate targets compared to IMiDs, resulting in enhanced anti-proliferative, pro-apoptotic and immune stimulatory activity. In early clinical studies, IBER has been shown to overcome resistance to IMiDs and in the ongoing Phase 3 EXCALIBER study (NCT04975997) is being explored for treatment of newly diagnosed (ND) and relapsed/refractory (RR) MM in combination with DARA and dexamethasone. Here, we investigate the potential mechanisms of synergy between IBER and DARA in preclinical models. Methods: Complement dependent cytotoxicity (CDC) of DARA was assessed with 15% human serum for human complement components and assays were performed by titrating either single agent or combinations in a tritiated thymidine assay. Antibody dependent cellular cytotoxicity (ADCC) was performed by co-culturing MM cells with varying expression levels of CD38 and effector cells and assessing target cell viability with annexin-v/topro-3 flow. A humanized PBMC adoptive transfer mouse model, utilizing human PBMC engraftment 14 days prior to implantation, was used to assess in vivo activity of IBER, DARA and IBER + DARA. Results: In CDC assays, we observed variable sensitivity of MM cells to complement-specific CDC in the presence of DARA (0.1-1 µg/ml), regardless of cell surface CD38 expression. Notably, IBER treatment resulted in upregulation of CD38 surface expression in 60% of MM cell lines tested. LP1 and MOLP8 displayed susceptibility to DARA-mediated CDC. DARA showed a dose-dependent increase in CDC activity in both cell lines. Notably, IBER+DARA also showed ~35% more synergistic CDC activity in both cell lines, than the single agents. In ADCC assays, we observed pretreatment of PBMC with IBER (vs. DMSO) significantly increased DARA mediated ADCC in H929 and LP-1 cells. To confirm that this effect was directed by NK cells, we cultured NK cells for 24 hr in supernatants from stimulated PBMC in presence of IBER prior to co-culture with MM cells. We noted that IBER-treated PBMC supernatant significantly enhanced the NK ADCC activity of DARA suggesting that IBER enhancement of cytokine secretion from PBMC increases NK cell mediated ADCC. Orthogonally, we pre-treated MM cells with IBER or DMSO prior to co-culture in ADCC assay. Notably, IBER treatment of MM cells, without treating immune cells, also increased DARA-mediated ADCC, indicating that IBER directly increases sensitivity to DARA-directed ADCC. To investigate the MOA of IBER-DARA synergy in ADCC, we analyzed gene expression and cell surface changes of a panel of NK-specific receptor ligands on MM cells following treatment with IBER. We observed IBER-induced upregulation of MICA mRNA levels (~2x) and surface expression (~40%) in MM target cells. We used RNAi to knock down Ikaros, an IBER substrate, which resulted in increased MICA expression (~3x) in MM cells. Furthermore, we observed that RNAi knock down of MICA in MM cells resulted in a significant decrease (60%, p<0.0001) in MM cell killing by NK cells, suggesting IBER-enhanced sensitivity of MM cells to NK-cell killing was in part, due to the upregulation of MICA through degradation of Ikaros. Lastly, we examined the anti-MM effects of IBER in a humanized xenograft model with a lenalidomide resistant cell line. Longitudinal analysis identified persistence of human PBMC engraftment and activation of immune subsets (NK cells and T-cells) that were increased upon IBER treatments, consistent with clinical observations. The combination of IBER/DARA resulted in ~22% tumor growth inhibition (TGI) in comparison to single agents, DARA and IBER and ~37% TGI in comparison to vehicle. Conclusion: Taken together, these data demonstrate that IBER and DARA work synergistically in preclinical models primarily through enhancement of both ADCC and CDC. This combination has the potential to improve outcomes in MM.

Development and Testing of a Glycoengineered Anti-ROR1 Antibody with Enhanced Capacity for Directing Antibody-Dependent Cellular Cytotoxicity (ADCC) of Chronic Lymphocytic Leukemia Cells

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic surface antigen expressed on the neoplastic cells of patients with chronic lymphocytic leukemia (CLL) or other types of cancer, but not on virtually all normal postpartum tissues (Fukuda T., et al., PNAS, 105:3047, 2008). Zilovertamab (also known as UC-961 or cirmtuzumab) is a humanized anti-ROR1 monoclonal antibody (mAb) currently under clinical investigation; this mAb is capable of blocking ROR1-signaling, which can contribute to cancer-cell migration, proliferation, survival, and self-renewal. We generated a glycoengineered form of zilovertamab (GE- zilovertamab) and examined whether it could mediate enhanced antibody-dependent cellular cytotoxicity (ADCC) against neoplastic cells that express ROR1. Initial studies used Jurkat-Lucia™ NFAT-CD16 cells (Invivogen, San Diego, CA, USA) which express high-affinity CD16A (FcγRIIIA, V158 allotype) as effector cells (EC), and the CLL-cell line MEC1, or MEC1 cells transduced to express ROR1 (MEC1-ROR1), as target cells (TC), which each also express CD20. Co-culture of Jurkat-Lucia cells for 6 hours with the anti-CD20 mAb rituximab and MEC1 or MEC1-ROR1 cells induced Jurkat-Lucia cells to express a luciferase reporter gene under the control of an ISG54 minimal promoter fused to six NFAT response elements; this endowed the Jurkat-Lucia cells with high luminescence activity that was not observed in co-cultures of EC and TC without added mAb. Neither zilovertamab nor GE-zilovertamab endowed Jurkat-Lucia cells with higher than background luminescence activity when co-cultured with MEC1 cells. However, GE-zilovertamab could induce high levels of luminescence activity in Jurkat-Lucia cells co-cultured with MEC1-ROR1 cells that was comparable to that of rituximab, and significantly greater than that of zilovertamab. We generated an orthogonal system with which to examine the relative ADCC activity of each of these mAb, using EC generated from the NK cell line, NK92, which we transduced with a lentivirus encoding a newly-generated high-affinity variant of CD16A (CD16v), which has been modified to block its cleavage by ADAM17. NK92 or NK92-CD16v were used as EC to effect cytolysis of Cr 51-labeled MEC1, MEC1-ROR1, or primary CLL cells in a chromium-release assay. Addition of rituximab to co-cultures of EC with Cr 51-labeled MEC1, MEC1-ROR1, or primary CLL caused mAb-dose-dependent ADCC of each of these TC by NK92-CD16v that was significantly greater than that by parental NK92 cells. Again, neither zilovertamab nor GE-zilovertamab could direct ADCC by NK92-CD16v or NK92 ADCC of MEC1 cells, which lack ROR1. However, GE-zilovertamab directed dose-dependent ADCC of MEC1-ROR1 or primary ROR1 + CLL cells by either NK92-CD16v or NK92 at levels comparable to that directed by rituximab for either EC population; such levels were significantly greater than that directed by zilovertamab. ROR1 + CLL cells harboring del(17p) or mutations in TP53 (del(17p)/m TP53) and/or that were resistant to targeted therapies (e.g., inhibitors of BTK or BCL2), were as susceptible to GE-zilovertamab-directed ADCC as were CLL cells without del(17p)/m TP53 from patients who had not had prior therapy. These data demonstrate that GE-zilovertamab can direct high-level ADCC lysis of ROR1-expressing neoplastic cells with greater activity than zilovertamab, encouraging development of clinical studies to evaluate GE-zilovertamab for therapy of patients with CLL or other ROR1-positive cancers.

IL-4 Acts at an Early Fate-Determining Junction in Hematopoiesis to Induce NK Cell Subsets Expressing Endogenous CD16

Human NK cells are known to contain subsets of CD56 +CD16 + and CD56 +CD16 - cells. The former directly attack target cells by an antibody-dependent cellular cytotoxicity (ADCC) various organs in the body, whereas the latter are mainly found in lymph nodes and activate inflammatory and immune responses by secreting IFNγ. However, despite their importance in the biological defence systems, whether and how these subsets emerge is not yet fully understood. In particular, several recent studies have challenged the conventional linear differentiation model which positioned CD56 +CD16 - cells as progenitors of CD56 +CD16 + cells. However, during the stepwise fate specification from immature multipotent progenitors to NK cells, it is still unknown at which stage the final CD16-expressing capacity is determined and what stimuli are involved in this decision. Through cell fate tracking in human HPC-derived differentiation, present study has demonstrated that fate specification into distinct progenitor cells specific for the two subsets occurs at a stage before the cells have yet to express CD56. Furthermore, it showed that IL-4 has a positive effect, both in bona fide and PSC-derived hematopoiesis, on the fate decision to progenitor cells from which CD56 +CD16 + NK cells emerge. While IL-4 was previously known to act on NK cells after CD56 expression, the present results show that IL-4 is also involved in future fate decisions at a more earlier progenitor cell stage than previous reports. We first identified three CD34 -CD56 - NK progenitor cell fractions, which were clearly distinguished due to the intensity of CD117 expression (CD34 -CD56 -CD117 ++, CD34 -CD56 -CD 117 +, CD34 -CD56 -CD117 -) in the differentiation pathway from lineage-negative cord blood progenitors. Tracking the cell fate of each fraction showed that they all produced CD56 + NK cells, but that CD16-expressing cells emerged predominantly from the CD34 -CD56 -CD117 + and CD34 -CD56 -CD117 - fractions at a rate of 15-30%. In contrast, NK cells originating from the CD34 -CD56 -CD117 ++ fraction hardly expressed CD16. Based on expression profile comparisons, we next identified IL-4 as a factor involved in progenitor cell fate decisions. IL-4 stimulation during early stage differentiation increased the CD34 -CD56 -CD117 + and CD34 -CD56 -CD117 - fractions, and conversely reduced the CD34 -CD56 -CD117 ++ cells. As a result, not only was the emergence efficiency of CD16-expressing NK cells increased, but their cytotoxic potential was also enhanced. During this process, IL-4 enhanced EOMES expression, which was in common with previous reports on T cells. Finally, we investigated whether NK cells derived from human pluripotent stem cells (hPSCs) also develop along the same trajectory as bona fide hematopoietic cells. Using a refined system of our previously reported NK cell differentiation cultures, we found that, as in cord blood, many hPSC strains produced all of three progenitor cell fractions, and both CD16 + and CD16 - NK cells appeared at the end of culture. In contrast, some strains produced only CD16 - NK cells, which were nearly deficient in the CD34 -CD56 -CD117 + fraction on the way of differentiation. Interestingly, stimulation with IL-4 significantly reduced the proportion of CD34 -CD56 -CD117 ++ progenitor cells from those strains (52.8±14.4% to 10.4 ±4.7%), which instead also produced CD34 -CD56 -CD117 + progenitors (13±3.4% to 68.1 ±8.3%) from which around 15% of CD16 + NK cells were eventually generated (Figure). Our results support a branching model in human NK cell differentiation, no matter whether they are derived from cord blood or hPSCs. We found that IL-4 stimulation accelerates specification into the progenitors capable of producing CD16 + NK cells in the future at a much earlier stage than previously thought. Furthermore, we showed that IL-4 could correct strain-dependent differentiation propensities of hPSCs to stably induce functional NK cells with high ADCC activity.

NK cell-induced damage to P.falciparum-infected erythrocytes requires ligand-specific recognition and releases parasitophorous vacuoles that are phagocytosed by monocytes in the presence of immune IgG.

Natural killer (NK) cells lyse virus-infected cells and transformed cells through polarized delivery of lytic effector molecules into target cells. We have shown that NK cells lyse Plasmodium falciparum-infected red blood cells (iRBC) via antibody-dependent cellular cytotoxicity (ADCC). A high frequency of adaptive NK cells, with elevated intrinsic ADCC activity, in people chronically exposed to malaria transmission is associated with reduced parasitemia and resistance to disease. How NK cells bind to iRBC and the outcome of iRBC lysis by NK cells has not been investigated. We applied gene ablation in inducible erythrocyte precursors and antibody-blocking experiments with iRBC to demonstrate a central role of CD58 and ICAM-4 as ligands for adhesion by NK cells via CD2 and integrin αMβ2, respectively. Adhesion was dependent on opsonization of iRBC by IgG. Live imaging and quantitative flow cytometry of NK-mediated ADCC toward iRBC revealed that damage to the iRBC plasma membrane preceded damage to P. falciparum within parasitophorous vacuoles (PV). PV were identified and tracked with a P.falciparum strain that expresses the PV membrane-associated protein EXP2 tagged with GFP. After NK-mediated ADCC, PV were either found inside iRBC ghosts or released intact and devoid of RBC plasma membrane. Electron microscopy images of ADCC cultures revealed tight NK-iRBC synapses and free vesicles similar in size to GFP+ PV isolated from iRBC lysates by cell sorting. The titer of IgG in plasma of malaria-exposed individuals that bound PV was two orders of magnitude higher than IgG that bound iRBC. This immune IgG stimulated efficient phagocytosis of PV by primary monocytes. The selective NK-mediated damage to iRBC, resulting in release of PV, and subsequent phagocytosis of PV by monocytes may combine for efficient killing and removal of intra-erythrocytic P.falciparum parasite. This mechanism may mitigate the inflammation and malaria symptoms during blood-stage P. falciparum infection.

Fc-mediated functions of nirsevimab complement direct respiratory syncytial virus neutralization but are not required for optimal prophylactic protection.

Nirsevimab is an extended half-life (M252Y/S254T/T256E [YTE]-modified) monoclonal antibody to the pre-fusion conformation of the respiratory syncytial virus (RSV) Fusion protein, with established efficacy in preventing RSV-associated lower respiratory tract infection in infants for the duration of a typical RSV season. Previous studies suggest that nirsevimab confers protection via direct virus neutralization. Here we use preclinical models to explore whether fragment crystallizable (Fc)-mediated effector functions contribute to nirsevimab-mediated protection. Nirsevimab, MEDI8897* (i.e., nirsevimab without the YTE modification), and MEDI8897*-TM (i.e., MEDI8897* without Fc effector functions) binding to Fc γ receptors (FcγRs) was evaluated using surface plasmon resonance. Antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent complement deposition (ADCD), and antibody-dependent cellular cytotoxicity (ADCC) were assessed through in vitro and ex vivo serological analyses. A cotton rat challenge study was performed with MEDI8897* and MEDI8897*-TM to explore whether Fc effector functions contribute to protection from RSV. Nirsevimab and MEDI8897* exhibited binding to a range of FcγRs, with expected reductions in FcγR binding affinities observed for MEDI8897*-TM. Nirsevimab exhibited in vitro ADNP, ADCP, ADCD, and ADCC activity above background levels, and similar ADNP, ADCP, and ADCD activity to palivizumab. Nirsevimab administration increased ex vivo ADNP, ADCP, and ADCD activity in participant serum from the MELODY study (NCT03979313). However, ADCC levels remained similar between nirsevimab and placebo. MEDI8897* and MEDI8897*-TM exhibited similar dose-dependent reduction in lung and nasal turbinate RSV titers in the cotton rat model. Nirsevimab possesses Fc effector activity comparable with the current standard of care, palivizumab. However, despite possessing the capacity for Fc effector activity, data from RSV challenge experiments illustrate that nirsevimab-mediated protection is primarily dependent on direct virus neutralization.

CD38 x ICAM-1 Bispecific Antibody Is a Novel Approach for Treating Multiple Myeloma and Lymphoma.

The cluster of differentiation 38 (CD38) is a well-validated target for treating multiple myeloma. Although anti-CD38 mAbs have demonstrated outstanding initial responses in patients with multiple myeloma, nearly all patients eventually develop resistance and relapse. In addition, currently approved CD38 targeting therapies have failed to show monotherapy efficacy in lymphomas, where CD38 expression is present but at lower levels. To effectively target CD38 on tumor cells, we generated an antibody-dependent cellular cytotoxicity (ADCC) enhanced bispecific CD38 x intercellular cell adhesion molecule 1 (ICAM-1) antibody, VP301. This bispecific antibody targets unique epitopes on CD38 and ICAM-1 on tumor cells with reduced red blood cell binding compared with the benchmark CD38 antibody daratumumab. VP301 demonstrated potent ADCC and antibody-dependent cellular phagocytosis activities on a selected set of myeloma and lymphoma cell lines even those with low CD38 expression. In an ex vivo drug sensitivity assay, we observed responses to VP301 in multiple myeloma primary samples from relapsed/refractory patients. Moreover, VP301 demonstrated potent tumor inhibition activities in in vivo myeloma and lymphoma models. Interestingly, combination of VP301 with the immunomodulatory drug, lenalidomide, led to synergistic antitumor growth activity in an in vivo efficacy study. In conclusion, the CD38 x ICAM-1 bispecific antibody VP301 demonstrated promising efficacy and specificity toward CD38+ and ICAM-1+ tumor cells and represents a novel approach for treating multiple myeloma and lymphoma.

Higher HIV-1 Env gp120-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) Activity Is Associated with Lower Levels of Defective HIV-1 Provirus.

A cure for HIV-1 (HIV) remains unrealized due to a reservoir of latently infected cells that persist during antiretroviral therapy (ART), with reservoir size associated with adverse health outcomes and inversely with time to viral rebound upon ART cessation. Once established during ART, the HIV reservoir decays minimally over time; thus, understanding factors that impact the size of the HIV reservoir near its establishment is key to improving the health of people living with HIV and for the development of novel cure strategies. Yet, to date, few correlates of HIV reservoir size have been identified, particularly in pediatric populations. Here, we employed a cross-subtype intact proviral DNA assay (CS-IPDA) to quantify HIV provirus between one- and two-years post-ART initiation in a cohort of Kenyan children (n = 72), which had a median of 99 intact (range: 0-2469), 1340 defective (range: 172-3.84 × 104), and 1729 total (range: 178-5.11 × 104) HIV proviral copies per one million T cells. Additionally, pre-ART plasma was tested for HIV Env-specific antibody-dependent cellular cytotoxicity (ADCC) activity. We found that pre-ART gp120-specific ADCC activity inversely correlated with defective provirus levels (n = 68, r = -0.285, p = 0.0214) but not the intact reservoir (n = 68, r = -0.0321, p-value = 0.800). Pre-ART gp41-specific ADCC did not significantly correlate with either proviral population (n = 68; intact: r = -0.0512, p-value = 0.686; defective: r = -0.109, p-value = 0.389). This suggests specific host immune factors prior to ART initiation can impact proviruses that persist during ART.