Antibody-dependent Cellular Cytotoxicity Responses Research Articles

HIV/HBV coinfection remodels the immune landscape and natural killer cell ADCC functional responses.

HBV and HIV coinfection is a common occurrence globally, with significant morbidity and mortality. Both viruses lead to immune dysregulation including changes in natural killer (NK) cells, a key component of antiviral defense and a promising target for HBV cure strategies. Here we used high-throughput single-cell analysis to explore the immune cell landscape in people with HBV mono-infection and HIV/HBV coinfection, on antiviral therapy, with emphasis on identifying the distinctive characteristics of NK cell subsets that can be therapeutically harnessed. Our data show striking differences in the transcriptional programs of NK cells. HIV/HBV coinfection was characterized by an over-representation of adaptive, KLRC2 -expressing NK cells, including a higher abundance of a chemokine-enriched ( CCL3/CCL4 ) adaptive cluster. The NK cell remodeling in HIV/HBV coinfection was reflected in enriched activation pathways, including CD3ζ phosphorylation and ZAP-70 translocation that can mediate stronger antibody-dependent cellular cytotoxicity responses and a bias toward chemokine/cytokine signaling. By contrast, HBV mono-infection imposed a stronger cytotoxic profile on NK cells and a more prominent signature of "exhaustion" with higher circulating levels of HBsAg. Phenotypic alterations in the NK cell pool in coinfection were consistent with increased "adaptiveness" and better capacity for antibody-dependent cellular cytotoxicity compared to HBV mono-infection. Overall, an adaptive NK cell signature correlated inversely with circulating levels of HBsAg and HBV-RNA in our cohort. This study provides new insights into the differential signature and functional profile of NK cells in HBV and HIV/HBV coinfection, highlighting pathways that can be manipulated to tailor NK cell-focused approaches to advance HBV cure strategies in different patient groups.

Human Cytomegalovirus mRNA-1647 Vaccine Candidate Elicits Potent and Broad Neutralization and Higher Antibody-Dependent Cellular Cytotoxicity Responses Than the gB/MF59 Vaccine.

MF59-adjuvanted gB subunit (gB/MF59) vaccine demonstrated approximately 50% efficacy against human cytomegalovirus (HCMV) acquisition in multiple clinical trials, suggesting that efforts to improve this vaccine design might yield a vaccine suitable for licensure. A messenger RNA (mRNA)-based vaccine candidate encoding HCMV gB and pentameric complex (PC), mRNA-1647, is currently in late-stage efficacy trials. However, its immunogenicity has not been compared to the partially effective gB/MF59 vaccine. We assessed neutralizing and Fc-mediated immunoglobulin G (IgG) effector antibody responses induced by mRNA-1647 in both HCMV-seropositive and -seronegative vaccinees from a first-in-human clinical trial through 1 year following third vaccination using a systems serology approach. Furthermore, we compared peak anti-gB antibody responses in seronegative mRNA-1647 vaccinees to that of seronegative gB/MF59 vaccine recipients. mRNA-1647 vaccination elicited and boosted HCMV-specific IgG responses in seronegative and seropositive vaccinees, respectively, including neutralizing and Fc-mediated effector antibody responses. gB-specific IgG responses were lower than PC-specific IgG responses. gB-specific IgG and antibody-dependent cellular phagocytosis responses were lower than those elicited by gB/MF59. However, mRNA-1647 elicited higher neutralization and antibody-dependent cellular cytotoxicity (ADCC) responses. Overall, mRNA-1647 vaccination induced polyfunctional and durable HCMV-specific antibody responses, with lower gB-specific IgG responses but higher neutralization and ADCC responses compared to the gB/MF59 vaccine. NCT03382405 (mRNA-1647) and NCT00133497 (gB/MF59).

Phase 1 Study of SC291, a Hypoimmune, Allogeneic CD19-Directed CAR T Cell Therapy for Relapsed/Refractory B-Cell Malignancies (ARDENT) - Initial Clinical Data

ARDENT is a phase 1 study evaluating safety and tolerability of SC291, a hypoimmune (HIP), allogeneic, CD19-directed CAR T cell therapy in subjects with Non-Hodgkin's Lymphoma and Chronic Lymphocytic Leukemia (CLL). SC291 is derived from healthy donor CD4+ and CD8+ T cells that are genetically engineered to disrupt function of CD3 and HLA class I/II, overexpress CD47, and express a CD19-directed CAR. A primary obstacle for allogeneic CAR T cell approaches remains their immune recognition and rejection. Patient immune responses prevent durable allogeneic CAR T clinical responses despite the use of enhanced lymphodepletion (LD) regimens. We therefore developed hypoimmune CAR T cells that avoid immune recognition and have the potential to deliver clinical efficacy while using a standard LD chemotherapy regimen (as defined by approved autologous CAR T products). The HIP engineering approach involves the depletion of HLA class I/II to prevent adaptive immune rejection and the overexpression of CD47 to prevent innate immune rejection, which has previously demonstrated alloimmune protection in preclinical models ( Nat Biotechnol 2019;37(3):252-258; Proc Natl Acad Sci U S A 2021;118(28):e2022091118; J Exp Med. 2021;218(3):e20200839; Nat Biotechnol. 2023; doi: 10.1038/s41587-023-01784-x, and Nat Commun. 2023;14(1):2020). The initial patient, a 74-year-old male with CLL (unmutated IGHV, del(11q), BTK C481S mutation) and 3 prior lines of therapy (FCR, ibrutinib, venetoclax + rituximab), received a LD regimen of cyclophosphamide 500 mg/m 2 and fludarabine 24 mg/m 2 (daily for 3 days) followed by a starting dose of 60 million CAR+ SC291 cells (of which approximately 80% are fully HIP engineered cells). SC291 was well tolerated with no observed CRS or ICANS. The patient developed non-neutropenic fever after LD (prior to SC291 infusion). At the Day 28 visit post-SC291 infusion, the patient had a partial response (per iwCLL 2018 criteria), along with significant (99%) B-cell reduction and improvements in platelet levels. Analysis of cellular kinetics is ongoing. SC291 is a mixture of T cells, in which portions are partially or fully HIP-engineered. As such, we evaluated the initial patient's immune response against SC291 subpopulations using a panel of previously described in vitro assays. Blood samples from the patient were taken pre-treatment (Day -5) and at the Day 13 and Day 28 visits following SC291 treatment and were assessed for immune evasion. The patient's PBMCs were sorted into CD3+ T cells and CD3-CD56+ NK cells and incubated with the following subpopulations sorted from SC291 drug product (DP): (i) fully HIP engineered (CD19 HIP CAR T cells), (ii) HLA I/II negative T cells without CD47 overexpression (DKO), and (iii) HLA I/II positive CAR T cells overexpressing CD47 (WT CD19 CAR T cells). Patient serum from Day -5 and the Day 28 visit were used for donor-specific antibody analyses, complement-dependent cytotoxicity (CDC), and antibody-dependent cellular cytotoxicity (ADCC) against the DP subpopulations. At Day -5, T cell EliSpot assays showed no immune responses against any of the SC291 sorted subpopulations, but by Days 13 and 28, these assays produced high and increasing IFN-g spot frequencies against the WT CD19 CAR T cells. Similar results were shown with T cell cytotoxicity assays (Fig. 1B). In contrast, no T cell activation and no T cell killing was observed with DKO and CD19 HIP CAR T cells at any time point. As expected, patient NK cells vigorously killed DKO T cells at all time points but spared WT CD19 CAR T and CD19 HIP CAR T cells (Fig. 1B). No antibodies against any sorted subpopulation were detected at Day -5, but IgG antibody binding of the WT CD19 CAR T population was observed at Day 28. These antibodies did not bind to DKO or CD19 HIP CAR T cells, and there was no CDC or ADCC response against these subpopulations. Interestingly, with the LD regimen used, we observed a 95% and 99% reduction of CD19+ cells on Days 0 and 28, respectively, while T cells rebounded from an 88% reduction on day 0 to pretreatment levels on Day 28 (Fig. 1A). These data demonstrate the preliminary safety and tolerability of SC291 in the initial treated patient. Immune assays demonstrated that the CD19 HIP CAR T cell subpopulation effectively evades the host adaptive and innate immune responses and could overcome the allogeneic barrier in humans. Additional data from the ARDENT study will be presented at the time of the conference.

Examining the impact of immunosuppressive drugs on antibody-dependent cellular cytotoxicity (ADCC) of human peripheral blood natural killer (NK) cells and gamma delta (γδ) T cells

BackgroundHuman natural killer (NK) cells and gamma delta (γδ) T cells may impact outcomes of solid organ transplantation (SOT) such as lung transplantation (LTx) following the differential engagement of an array of activating and inhibitory receptors. Amongst these, CD16 may be particularly important due to its capacity to bind IgG to trigger antibody-dependent cellular cytotoxicity (ADCC) and the production of proinflammatory cytokines. While the use of immunosuppressive drugs (ISDs) is an integral component of SOT practice, their relative impact on various immune cells, especially γδT cells and CD16-induced functional responses, is still unclear. MethodsThe ADCC responses of peripheral blood NK cells and γδT cells from both healthy blood donors and adult lung transplant recipients (LTRs) were assessed by flow cytometry. Specifically, the degranulation response, as reflected in the expression of CD107a, and the capacity of both NK cells and γδT cells to produce IFN-γ and TNF-α was assessed following rituximab (RTX)-induced activation. Additionally, the effect of cyclosporine A (CsA), tacrolimus (TAC), prednisolone (Prdl) and azathioprine (AZA) at the concentration of 1 ng/ml, 10 ng/ml, 100 ng/ml, and 1000 ng/ml on these responses was also compared in both cell types. ResultsFlow cytometric analyses of CD16 expresion showed that its expression on γδT cells was both at lower levels and more variable than that on peripheral blood NK cells. Nevertheless functional analyses showed that despite these differences, γδT cells like NK cells can be readily activated by engagement with RTX to degranulate and produce cytokines such as IFNg and TNF-a. RTX-induced degranulation by either NK cells or γδT cells from healthy donors was not impacted by co-culture with individual ISDs. However, CsA and TAC but not Prdl and AZA did inhibit the production of IFN-γ and TNF-α by both cell types. Flow cytometric analyses of RTX-induced activation of NK cells and γδT cells from LTRs suggested their capacity to degranulate was not markedly impacted by transplantation with similar levels of cells expressing CD107 pre- and post-LTx. However an impairment in the ability of NK cells to produce cytokines was observed in samples obtained post LTx whereas γδT cell cytokine responses were not significantly impacted. ConclusionsIn conclusion, the findings show that despite differences in the expression levels of CD16, γδT cells like NK cells can be readily activated by engagement with RTX and that in vitro exposure to CsA and TAC (calcineurin inhibitors) had a measurable effect on cytokine production but not degranulation by both NK cells and gdT cells from healthy donors. Finally the observation that in PBMC obtained from LTx recipients, NK cells but not γδT cells exhibited impaired cytokine reponses suggests that transplantation or chronic exposure to ISDs differentially impacts their potential to respond to the introduction of an allograft and/or transplant-associated infections.

Oral Immunization with rVSV Bivalent Vaccine Elicits Protective Immune Responses, Including ADCC, against Both SARS-CoV-2 and Influenza A Viruses.

COVID-19 and influenza both cause enormous disease burdens, and vaccines are the primary measures for their control. Since these viral diseases are transmitted through the mucosal surface of the respiratory tract, developing an effective and convenient mucosal vaccine should be a high priority. We previously reported a recombinant vesicular stomatitis virus (rVSV)-based bivalent vaccine (v-EM2/SPΔC1Delta) that protects animals from both SARS-CoV-2 and influenza viruses via intramuscular and intranasal immunization. Here, we further investigated the immune response induced by oral immunization with this vaccine and its protective efficacy in mice. The results demonstrated that the oral delivery, like the intranasal route, elicited strong and protective systemic immune responses against SARS-CoV-2 and influenza A virus. This included high levels of neutralizing antibodies (NAbs) against SARS-CoV-2, as well as strong anti-SARS-CoV-2 spike protein (SP) antibody-dependent cellular cytotoxicity (ADCC) and anti-influenza M2 ADCC responses in mice sera. Furthermore, it provided efficient protection against challenge with influenza H1N1 virus in a mouse model, with a 100% survival rate and a significantly low lung viral load of influenza virus. All these findings provide substantial evidence for the effectiveness of oral immunization with the rVSV bivalent vaccine.

Impact of HIV-1 Vpu-mediated downregulation of CD48 on NK-cell-mediated antibody-dependent cellular cytotoxicity.

HIV-1 evades antibody-dependent cellular cytotoxicity (ADCC) responses not only by controlling Env conformation and quantity at the cell surface but also by altering NK cell activation via the downmodulation of several ligands of activating and co-activating NK cell receptors. The signaling lymphocyte activation molecule (SLAM) family of receptors, which includes NTB-A and 2B4, act as co-activating receptors to sustain NK cell activation and cytotoxic responses. These receptors cooperate with CD16 (FcγRIII) and other activating receptors to trigger NK cell effector functions. In that context, Vpu-mediated downregulation of NTB-A on HIV-1-infected CD4 T cells was shown to prevent NK cell degranulation via an homophilic interaction, thus contributing to ADCC evasion. However, less is known on the capacity of HIV-1 to evade 2B4-mediated NK cell activation and ADCC. Here, we show that HIV-1 downregulates the ligand of 2B4, CD48, from the surface of infected cells in a Vpu-dependent manner. This activity is conserved among Vpu proteins from the HIV-1/SIVcpz lineage and depends on conserved residues located in its transmembrane domain and dual phosphoserine motif. We show that NTB-A and 2B4 stimulate CD16-mediated NK cell degranulation and contribute to ADCC responses directed to HIV-1-infected cells to the same extent. Our results suggest that HIV-1 has evolved to downmodulate the ligands of both SLAM receptors to evade ADCC. IMPORTANCE Antibody-dependent cellular cytotoxicity (ADCC) can contribute to the elimination of HIV-1-infected cells and HIV-1 reservoirs. An in-depth understanding of the mechanisms used by HIV-1 to evade ADCC might help develop novel approaches to reduce the viral reservoirs. Members of the signaling lymphocyte activation molecule (SLAM) family of receptors, such as NTB-A and 2B4, play a key role in stimulating NK cell effector functions, including ADCC. Here, we show that Vpu downmodulates CD48, the ligand of 2B4, and this contributes to protect HIV-1-infected cells from ADCC. Our results highlight the importance of the virus to prevent the triggering of the SLAM receptors to evade ADCC.

AB-101, an allogeneic, non–genetically modified, natural killer (NK) cell therapy, evaluated as monotherapy or in combination with rituximab in R/R non-Hodgkin lymphoma.

7529 Background: AB-101 is a non-genetically modified, cord blood-derived, allogeneic, cryopreserved, NK cell therapy for use in combination with monoclonal antibody (mAb) or innate cell engager therapies. AB-101 is being developed to enhance response to mAb therapy through the antibody-dependent cellular cytotoxicity (ADCC) mechanism. Many cancer patients (pts) are unable to mount a robust ADCC response, rendering mAb therapy less effective for these pts. AB-101 has attributes that enhance NK cell activity: Consistent phenotype for tumor engagement and cytotoxicity; Selected killer immunoglobulin-like receptor B (KIR B) haplotype; V/V CD16 polymorphism at F158 for enhanced ADCC. AB-101 outpatient administration provides optimized NK cells to pts, potentially enhancing their anti-tumor activity in combination with mAb that utilize the ADCC mechanism. We have initiated a Ph 1/2 clinical trial evaluating the combination of AB-101 with rituximab (RTX) in pts with R/R NHL of B-cell origin. Methods: Two AB-101 dose levels, 1 or 4e9 cells, will be evaluated as monotherapy or combination with RTX. All enrolled pts will receive at least 1 cycle of 4 weekly AB-101 doses, with cyclophosphamide/fludarabine lymphodepletion (LD). Pts receiving the combination may receive up to 4 treatment cycles. Results: Dose escalation is ongoing with 17 pts enrolled. Pts were elderly (median of 68 yrs) and heavily pretreated (median of 4 prior lines) with 64% having had prior CD19 CAR-T. Pts had DLBCL (9), FL (5), MCL (2), and LPL/WM (1). 11 monotherapy pts and 6 RTX combination pts were treated at time of abstract. 16 pts received AB-101 at 1e9 cells/dose; 1 pt received 4e9 cells/dose. 4 of 6 combination pts have received > 1 cycle. AB-101 was generally well tolerated. The most common G3+ AEs were LD-related neutropenia and lymphopenia. All AB-101 related AEs were G1/2. ICANs and GVHD, events associated with allogeneic cell therapy, were not reported; Gr 1 CRS occurred in 2 pts and resolved within 24h. SAEs were reported in 9 pts. The most common SAEs were associated with either infections (n = 3) or PD (n = 3) and one related SAE of Gr 1 fever was reported. No pts discontinued treatment due to an AB-101-related AE. ORR for the RTX cohort was 67% (4/6) with a CR rate of 50% (3/6). 3 of 4 responses were observed in pts who failed prior CAR-T therapy. ORR for the monotherapy cohorts was 27% (3/11). Conclusions: 4 weekly doses of AB-101 given alone or with RTX appeared to be safe and well tolerated without the serious/severe toxicities associated with CAR-T therapies. Preliminary efficacy of the combination treatment is encouraging. The study is ongoing and continues to enroll the combination cohorts and the 4e9 cells/dose level. Updated safety, efficacy, and exploratory PK/PD/immunogenicity results will be provided during the presentation. Clinical trial information: NCT04673617 .

Reconstruction of a polyclonal ADCC antibody repertoire from an HIV-1 non-transmitting mother

Human natural history and vaccine studies support a protective role of antibody dependent cellular cytotoxicity (ADCC) activity against many infectious diseases. One setting where this has consistently been observed is in HIV-1 vertical transmission, where passively acquired ADCC activity in HIV-exposed infants has correlated with reduced acquisition risk and reduced pathogenesis in HIV+ infants. However, the characteristics of HIV-specific antibodies comprising a maternal plasma ADCC response are not well understood. Here, we reconstructed monoclonal antibodies (mAbs) from memory B cells from late pregnancy in mother MG540, who did not transmit HIV to her infant despite several high-risk factors. Twenty mAbs representing 14 clonal families were reconstructed, which mediated ADCC and recognized multiple HIV Envelope epitopes. In experiments using Fc-defective variants, only combinations of several mAbs accounted for the majority of plasma ADCC of MG540 and her infant. We present these mAbs as evidence of a polyclonal repertoire with potent HIV-directed ADCC activity.

Temsavir blocks the immunomodulatory activities of HIV-1 soluble gp120

While HIV-1-mediated CD4 downregulation protects infected cells from antibody-dependent cellular cytotoxicity (ADCC), shed gp120 binds to CD4 on uninfected bystander CD4+ Tcells, sensitizing them to ADCC mediated by HIV+ plasma. Soluble gp120-CD4 interaction on multiple immune cells also triggers a cytokine burst. The small molecule temsavir acts as an HIV-1 attachment inhibitor by preventing envelope glycoprotein (Env)-CD4 interaction and alters the overall antigenicity of Env by affecting its processing and glycosylation. Here we show that temsavir also blocks the immunomodulatory activities of shed gp120. Temsavir prevents shed gp120 from interacting with uninfected bystander CD4+ cells, protecting them from ADCC responses and preventing a cytokine burst. Mechanistically, this depends on temsavir's capacity to prevent soluble gp120-CD4 interaction, to reduce gp120 shedding, and to alter gp120 antigenicity. This suggests that the clinical benefits provided by temsavir could extend beyond blocking viral entry.

The human cytomegalovirus-specific and UL40-mediated imprint in the natural killer cell repertoire is associated with antibody-mediated rejection in lung transplant recipients.

CD16+ natural killer (NK-) cells play, together with donor-specific antibodies (DSA) and via antibody-dependent cellular cytotoxicity (ADCC), an important role in the pathogenesis of antibody-mediated rejection (ABMR) in lung-transplant recipients (LTRs). Cytotoxic CD16+NKG2C+ NK cells proliferate in response to human Cytomegalovirus (HCMV) infections via the presentation of HCMV-encoded and highly polymorphic UL40 peptides. In our study, we aimed to clarify whether infections with HCMV-strains carrying different UL40 peptide variants are associated with the shift of the NK cell repertoire and the development of ABMR in LTRs. We included 30 DSA+ABMR+, 30 DSA+ABMR- and 90 DSA-ABMR- LTRs. In all patients, 1 episode of high-level HCMV-replication occurred. In all DSA+ABMR+ LTRs, HCMV-replication occurred prior to ABMR diagnosis. The association of HCMV UL40 variants with the expansion of CD16+ NK cell subsets and ABMR was assessed in NK cell proliferation and ADCC assays. Our study revealed that the VMAPRTLIL and VMTPRTLVL UL40 variants were significantly overrepresented in DSA+ABMR+ LTRs. Both peptides were associated with a pronounced proliferation of cytotoxic and proinflammatory CD16+NKG2C+ NK cells. The stimulation with both peptides led to a shift of the NK cell repertoire towards CD16+NKG2C+ NK cells, which was associated with strong ADCC responses after stimulation with endothelial cells and plasma from DSA+ABMR+ LTRs. Distinct UL40 peptide variants of the infecting HCMV-strain are associated with the development of ABMR after lung transplantation, due to a shift towards a highly cytotoxic CD16+NKG2C+ NK cell population. These peptides are thus potential prognostic markers for ABMR.

Pre-Clinical Efficacy of AB-101, an Allogeneic Cord-Blood Derived Natural Killer (NK) Cell Therapeutic Candidate, in Combination with Anti-CD38 Antibodies in Models of Multiple Myeloma

Introduction: AB-101 is a non-engineered, allogeneic, off-the-shelf, cryopreserved cord blood-derived natural killer (NK)-cell therapy in development as a cancer therapeutic. A highly scaled manufacturing process enables production of 1000s of doses of AB-101 from a single donor cord blood unit (CBU). AB-101 is derived from CBUs selected for KIR-B haplotype and natural high-affinity variant of CD16 (158V/V), which are associated with improved anti-tumor activity and ADCC enhancement, resulting in a highly active NK cell product without the requirement for additional engineering. We are developing AB-101 for combination with monoclonal antibodies (mAbs) to enhance antibody dependent cellular cytotoxicity (ADCC) in patients, initially in an ongoing Phase I clinical trial for NHL. To further develop AB-101 as an ADCC enhancer, we assessed anti-tumor activity in combination with CD38 mAb, against multiple myeloma (MM) cell lines. CD38 is a transmembrane glycoprotein that is uniformly and highly upregulated on MM cells. Anti-CD38 mAbs exert anti-tumor activity against MM through ADCC and other mechanisms. NK cells play a key role in ADCC responses through CD16 receptor signaling. However, the majority of NK cells express CD38 and are depleted by therapeutic CD38 mAbs which could limit their therapeutic efficacy. Combining NK cell therapies with anti-CD38 mAbs, thus necessitates CD38 knockout to prevent fratricide. AB-101 has a naturally low percent and intensity of CD38 expression. Here we present data demonstrating that AB-101 are resistant to anti-CD38 mAb-induced fratricide and can elicit significant ADCC against MM cell lines both in vitro and in vivo. Since anti-CD38 mAbs are combined with glucocorticoids in the clinic, we assessed the impact of dexamethasone and methylprednisolone on ADCC by AB-101. Here we report robust ADCC following treatment of AB-101 with glucocorticoids. Methods: AB-101 was screened for CD38 expression and incubated with anti-CD38 mAb - daratumumab or hIgG1 control (300 - 0.41 µg/ml) for 4 hours to assess fratricide. In vitro ADCC assays were performed at various effector to target ratios against control (Daudi and K562) and MM cell lines (NCI-H929, RPMI 8226, MM.1S and MM.1R) using 2 µg/ml of daratumumab or hIgG1. To study the effect of glucocorticoids, AB-101 was cultured in various concentrations of dexamethasone and methylprednisolone for 48 hours in the presence of 50 or 500 IU/ml of IL-2, prior to ADCC assay. In vivo efficacy of AB-101 in combination with daratumumab was assessed in the intraperitoneal (IP) MM.1S-Luc xenograft model in NSG mice (Figure 1A). Daratumumab and AB-101 were administered by IP injection. Tumor burden was assessed by bioluminescent imaging (BLI). Results: AB-101 was over 98% CD56+/CD3- with 87% CD56+/CD16+ population. Only 22% of GMP drug product were CD38+ with low intensity of expression compared to peripheral blood NK cells (~88%). In the presence of daratumumab AB-101 viability was unaltered compared to hIgG1 control demonstrating resistance to fratricide. Daratumumab enhanced ADCC potential of AB-101 against MM cell lines and Daudi in in vitro. ADCC against MM lines and Daudi did not decrease on exposure to glucocorticoids compared to no treatment group. In vivo anti-tumor efficacy, as evidenced by reduction in BLI signal, was observed in the MM.1S model in AB-101 + daratumumab groups compared to daratumumab and AB-101 alone (Figure 1B). AB-101 + daratumumab (1 and 3mpk) led to 98 and 99% tumor growth inhibition (TGI), respectively, relative to no treatment. Daratumumab monotherapy at 1 and 3mpk showed 21% and 87% TGI respectively, and AB-101 showed 16% TGI relative to no treatment. Conclusions: AB-101 has high and consistent expression of CD16 and low CD38 expression and is resistant to daratumumab induced fratricide and hence can be combined with daratumumab without additional engineering such as CD38 knockout. Daratumumab is combined in the clinic with glucocorticoids, immunomodulatory drugs (IMiDs) and proteosome inhibitors (PI). While IMiDs and PIs are reported to enhance NK cell function, glucocorticoids have been shown to diminish NK cell cytotoxicity. Data presented here suggests that ADCC potential of AB-101 is not negatively impacted by glucocorticoids. Taken together, the in vitro and in vivo data supports further clinical development of AB-101 in combination with daratumumab to treat MM patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Low quantity and quality of anti-spike humoral response is linked to CD4 T-cell apoptosis in COVID-19 patients

In addition to an inflammatory reaction, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-infected patients present lymphopenia, which we recently reported as being related to abnormal programmed cell death. As an efficient humoral response requires CD4 T-cell help, we hypothesized that the propensity of CD4 T cells to die may impact the quantity and quality of the humoral response in acutely infected individuals. In addition to specific immunoglobulins (Ig)A, IgM, and IgG against SARS-CoV-2 nucleocapsid (N), membrane (M), and spike (S1) proteins, we assessed the quality of IgG response by measuring the avidity index. Because the S protein represents the main target for neutralization and antibody-dependent cellular cytotoxicity responses, we also analyzed anti-S-specific IgG using S-transfected cells (S-Flow). Our results demonstrated that most COVID-19 patients have a predominant IgA anti-N humoral response during the early phase of infection. This specific humoral response preceded the anti-S1 in time and magnitude. The avidity index of anti-S1 IgG was low in acutely infected individuals compared to convalescent patients. We showed that the percentage of apoptotic CD4 T cells is inversely correlated with the levels of specific IgG antibodies. These lower levels were also correlated positively with plasma levels of CXCL10, a marker of disease severity, and soluble Fas ligand that contributes to T-cell death. Finally, we found lower S-Flow responses in patients with higher CD4 T-cell apoptosis. Altogether, these results demonstrate that individuals with high levels of CD4 T-cell apoptosis and CXCL10 have a poor ability to build an efficient anti-S response. Consequently, preventing CD4 T-cell death might be a strategy for improving humoral response during the acute phase, thereby reducing COVID-19 pathogenicity.

Antibody-dependent cellular cytotoxicity responses and susceptibility influence HIV-1 mother-to-child transmission.

HIV-1 vaccine efforts are primarily directed toward eliciting neutralizing antibodies (nAbs). However, vaccine trials and mother-to-child natural history cohort investigations indicate that antibody-dependent cellular cytotoxicity (ADCC), not nAbs, correlate with prevention. The ADCC characteristics associated with lack of HIV-1 acquisition remain unclear. Here, we examine ADCC and nAb properties in pretransmission plasma from HIV-1–exposed infants and from the corresponding transmitting and nontransmitting mothers’ breast milk and plasma. Breadth and potency (BP) were assessed against a panel of heterologous, nonmaternal variants. ADCC and neutralization sensitivity were estimated for the strains in the infected mothers. Infants who eventually acquired HIV-1 and those who remained uninfected had similar pretransmission ADCCBP. Viruses circulating in the transmitting and nontransmitting mothers had similar ADCC susceptibility. Infants with higher pretransmission ADCCBP and exposure to more ADCC-susceptible strains were less likely to acquire HIV-1. In contrast, higher preexisting infant neutralization BP and greater maternal virus neutralization sensitivity did not associate with transmission. Infants had higher ADCCBP closer to birth and in the presence of high plasma IgG relative to IgA levels. Mothers with potent humoral responses against their autologous viruses harbored more ADCC-sensitive strains. ADCC sensitivity of the exposure variants and preexisting ADCCBP influenced mother-to-child HIV-1 transmission during breastfeeding. Vaccination strategies that enhance ADCC are likely insufficient to prevent HIV-1 transmission because some strains may have low ADCC susceptibility.

High-affinity FcγRIIIa genetic variants and potent NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) responses contributing to severe COVID-19

PurposeHost genetic variants in activating natural killer (NK) cell receptors may contribute to differences in severity of COVID-19. NK cell-mediated antibody-mediated cellular cytotoxicity (ADCC) responses play, however, a controversial role in SARS-CoV-2 infections. It is unclear whether proinflammatory and cytotoxic SARS-CoV-2-specific ADCC responses limit disease severity or rather contribute to the immunopathogenesis of severe COVID-19. MethodsUsing a genetic association approach and subsequent in vitro antibody-dependent NK cell activation experiments, we investigated whether genetic variants in the FcγRIIIa-encoding FCGR3A gene, resulting in expression of either a low-affinity or high-affinity variant, and individual SARS-CoV-2-specific ADCC response contribute to COVID-19 severity. ResultsIn our study, we showed that the high-affinity variant of the FcγRIIIa receptor, 158-V/V, is significantly over-represented in hospitalized and deceased patients with COVID-19, whereas the low-affinity FcγRIIIa-158-F/F variant occurs more frequently in patients with mild COVID-19 (P < .0001). Furthermore, functional SARS-CoV-2 antibody-specific NK cell-mediated ADCC assays revealed that significantly higher proinflammatory ADCC responses occur in hospitalized patients with COVID-19, and are especially observed in NK cells expressing the FcγRIIIa-158-V/V variant (P < .0001). ConclusionOur study provides evidence that pronounced SARS-CoV-2-specific NK cell-mediated ADCC responses are influenced by NK cell FcγRIIIa genetic variants and are a hallmark of severe COVID-19.

Potential of Antibody-Dependent Cellular Cytotoxicity in Acute and Recovery Phases of SARS-CoV-2 Infection

Abstract Background: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused a global pandemic that has resulted in millions of casualties. Although researchers have reported the existence of neutralizing antibodies and viral T cell immunity against SARS-CoV-2, little is known about the presence of antibody-dependent cellular cytotoxicity (ADCC) and its role in combating SARS-CoV-2 infection. Methods: Nineteen acute COVID-19 patients at the First Affiliated Hospital of Guangzhou Medical University from January to February, 2020 and 55 recovery COVID-19 patients at the Second Peoples Hospital of Changde City from February, 2020 to February, 2021 were recruited in this study. Longitudinal plasma samples were collected. A virus-specific ADCC assay was performed to study the COVID-19 plasma samples. The correlations between ADCC and total IgG titer, including anti-RBD, anti-N, and neutralizing antibody titer were analyzed. Results: A high level of ADCC with 0.86% of IFN-γ+CD107a+ NK cells induced by anti RBD antibodies and with 0.54% of IFN-γ+CD107a+ NK cells induced by anti N antibodies was observed. This activity peaked at 3 weeks after disease onset with 1.16% and 0.63% of IFN-γ+CD107a+ NK cells induced by anti RBD and anti N antibodies respectively, declined to 0.32% and 0.32% of IFN-γ+CD107a+ NK cells respectively after more than 2 months, and persisted for 12 months after disease onset. The ADCC did not aggravate the severity of COVID-19 in terms of sequential organ failure assessment, although ADCC decreased with the age of COVID-19 patients. Interestingly, ADCC response is not correlated with neutralizing antibody titer or total IgG titers against S protein RBD and N protein in acute patients. ADCC in recovered patients showed a significant correlation with anti RBD IgG titer (R 2 = 0.33, P &lt; 0.001). Conclusion: Antibodies from COVID-19 patients against the N protein and S protein RBD domains could stimulate high levels of ADCC response. Our results provide evidence that vaccination should not only focus on neutralizing antibodies but also binding antibodies that may facilitate the antiviral function of ADCC, especially in the elderly.

Natural Killer Cell-Mediated Antibody-Dependent Cellular Cytotoxicity Against SARS-CoV-2 After Natural Infection Is More Potent Than After Vaccination.

We compared the ability of SARS-CoV2 Spike-specific antibodies to induce natural killer (NK) cell-mediated antibody dependent cellular cytotoxicity (ADCC) in patients with natural infection and vaccinated persons. Analyzing plasma samples from 39 COVID-19 patients and 11 vaccinated individuals, significant induction of ADCC could be observed over a period of more than three months in both vaccinated and recovered individuals. Although plasma antibody concentrations were lower in recovered patients, we found antibodies elicited by natural infection induced a significantly stronger ADCC response compared to those induced by vaccination, which may affect protection conferred by vaccination.

Broadly binding and functional antibodies and persisting memory B cells elicited by HIV vaccine PDPHV

Since publishing our original reports on the safety and immunogenicity of a polyvalent DNA prime-protein boost HIV vaccine (PDPHV) which elicited high titer antibody responses with broad specificity, neutralizing activities to multiple HIV-1 subtypes, as well as poly-functional T cell responses, accumulated findings from other HIV vaccine studies indicated the important roles of Ig isotype distribution, Fc medicated functions and the persistence of memory immune responses which were not studied in previous PDPHV related reports. The current report provides further detailed characterization of these parameters in human volunteers receiving the PDPHV regimen. Antibody responses were assessed using IgG isotype and gp70-V1V2-binding ELISAs, peptide arrays, and antibody-dependent cellular cytotoxicity (ADCC) assays. B cell ELISPOT was used to detect gp120-specific memory B cells. Our results showed that the gp120-specific antibodies were primarily of the IgG1 isotype. HIV-1 envelope protein variable regions V1 and V2 were actively targeted by the antibodies as determined by specific binding to both peptide and V1V2-carrying scaffolds. The antibodies showed potent and broad ADCC responses. Finally, the B cell ELISPOT analysis demonstrated persistence of gp120-specific memory B cells for at least 6 months after the last dose. These data indicate that broadly reactive binding Abs and ADCC responses as well as durable gp120-specific memory B cells were elicited by the polyvalent heterologous prime-boost vaccination regimens and showed great promise as a candidate HIV vaccine.

ADCC-mediating non-neutralizing antibodies can exert immune pressure in early HIV-1 infection.

Despite antibody-dependent cellular cytotoxicity (ADCC) responses being implicated in protection from HIV-1 infection, there is limited evidence that they control virus replication. The high mutability of HIV-1 enables the virus to rapidly adapt, and thus evidence of viral escape is a very sensitive approach to demonstrate the importance of this response. To enable us to deconvolute ADCC escape from neutralizing antibody (nAb) escape, we identified individuals soon after infection with detectable ADCC responses, but no nAb responses. We evaluated the kinetics of ADCC and nAb responses, and viral escape, in five recently HIV-1-infected individuals. In one individual we detected viruses that escaped from ADCC responses but were sensitive to nAbs. In the remaining four participants, we did not find evidence of viral evolution exclusively associated with ADCC-mediating non-neutralizing Abs (nnAbs). However, in all individuals escape from nAbs was rapid, occurred at very low titers, and in three of five cases we found evidence of viral escape before detectable nAb responses. These data show that ADCC-mediating nnAbs can drive immune escape in early infection, but that nAbs were far more effective. This suggests that if ADCC responses have a protective role, their impact is limited after systemic virus dissemination.

HIV-1 Envelope Glycoproteins Proteolytic Cleavage Protects Infected Cells from ADCC Mediated by Plasma from Infected Individuals.

The HIV-1 envelope glycoprotein (Env) is synthesized in the endoplasmic reticulum as a trimeric gp160 precursor, which requires proteolytic cleavage by a cellular furin protease to mediate virus-cell fusion. Env is conformationally flexible but controls its transition from the unbound “closed” conformation (State 1) to downstream CD4-bound conformations (States 2/3), which are required for fusion. In particular, HIV-1 has evolved several mechanisms that reduce the premature “opening” of Env which exposes highly conserved epitopes recognized by non-neutralizing antibodies (nnAbs) capable of mediating antibody-dependent cellular cytotoxicity (ADCC). Env cleavage decreases its conformational transitions favoring the adoption of the “closed” conformation. Here we altered the gp160 furin cleavage site to impair Env cleavage and to examine its impact on ADCC responses mediated by plasma from HIV-1-infected individuals. We found that infected primary CD4+ T cells expressing uncleaved, but not wildtype, Env are efficiently recognized by nnAbs and become highly susceptible to ADCC responses mediated by plasma from HIV-1-infected individuals. Thus, HIV-1 limits the exposure of uncleaved Env at the surface of HIV-1-infected cells at least in part to escape ADCC responses.

Pre-existing infant antibody-dependent cellular cytotoxicity associates with reduced HIV-1 acquisition and lower morbidity

SummaryIn humans, pre-existing anti-HIV-1 neutralizing antibodies (nAbs) have not been associated with decreased HIV-1 acquisition. Here, we evaluate antibody-dependent cellular cytotoxicity (ADCC) present in pre-transmission infant and maternal plasma and breast milk (BM) against the contemporaneous maternal HIV-1 variants. HIV-1-exposed uninfected compared with HIV-1-exposed infected infants have higher ADCC and a combination of ADCC and nAb responses against their corresponding mother’s strains. ADCC does not correlate with nAbs, suggesting they are independent activities. The infected infants with high ADCC compared with low ADCC, but not those with higher ADCC plus nAbs, have lower morbidity up to 1 year after birth. A higher IgA to IgG ratio, observed in BM supernatants and in a higher proportion of the infected compared with the uninfected infants, associates with lower ADCC. Against the exposure strains, ADCC, more than nAbs, associates with both lower mother-to-child transmission and decreased post-infection infant morbidity.