Antibody-dependent Cellular Phagocytosis Research Articles

Hemagglutinin stalk-specific Fc-mediated functions are associated with protection against influenza-illness after seasonal influenza vaccination in pregnant women.

Several influenza vaccine candidates aim to elicit antibodies against the conserved hemagglutinin stalk domain. Understanding the protective mechanism of these antibodies, which mediate broad neutralization and Fc-mediated functions, following seasonal vaccination is critical. Plasma samples were obtained from a subset of pregnant women living with or without HIV-1 enrolled in a randomised trial (138 trivalent inactivated vaccine [TIV] and 145 placebo recipients). Twenty-three influenza-illness cases were confirmed within 6 months postpartum. We measured H1 stalk-specific antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD) and cellular cytotoxicity (ADCC) at enrolment and 1-month post-vaccination. The association between these Fc-mediated functions and protection against influenza-illness following vaccination was examined using multiple logistic regression analysis and risk reduction thresholds were defined by the score associated with the lowest odds of influenza-illness. Amongst TIV and placebo recipients, lower H1 stalk-specific ADCP and ADCD activity was detected for participants with confirmed influenza compared with individuals without confirmed influenza-illness 1-month post-vaccination. Pre-existing ADCP scores ≥250 reduced the odds of A/H1N1 infection (odds ratio 0.11; p=0.01) with an 83% likelihood of risk reduction. Following TIV, ADCD scores of ≥25 and ≥15 significantly reduced the odds against A/H1N1 (0.10; p=0.01) and non-group 1 (0.06; p=0.0004) influenza virus infections, respectively. These ADCD scores were associated with >84% likelihood of risk reduction. H1 stalk-specific ADCC potential was not associated with protection against influenza-illness. H1 stalk-specific ADCD correlates with protection against influenza-illness following influenza vaccination during pregnancy. These findings provide insight into the protective mechanisms of HA stalk antibodies.

Druggability properties of a L309K mutation in the antibody CH2 domain.

In the early stages of antibody drug development, it is imperative to conduct a comprehensive assessment and enhancement of the druggability attributes of potential molecules by considering their fundamental physicochemical properties. This study specifically concentrates on the surface-exposed hydrophobic region of the candidate antibody aPDL1-WT and explores the effectiveness of the L309K mutation strategy. The resulting aPDL1-LK variant demonstrates a notable enhancement over the original antibody in addressing the issue of aggregation and formation of large molecular impurities under accelerated high-temperature conditions. The mutated molecule, aPDL1-LK, exhibits excellent physicochemical properties such as hydrophilicity, conformational stability, charge variant stability, post-translational modifications, and serum stability. In terms of biological function, aPDL1-LK maintains the same glycosylation pattern as the original antibody and shows no significant difference in affinity for antigen hPDL1 protein, CD16a-F158, CD64, CD32a-H131, and complement C1q, compared to aPDL1-WT. The L309K mutation results in an approximately twofold reduction in its affinity for CD16a-V158 and CD32a-R131. In vitro biological assays, including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), reveal that the L309K mutation may decrease CD16a-V158-mediated ADCC activity due to the mutation-induced decrease in ligand affinity, while not affect CD32a-R131-mediated ADCP activity. In conclusion, the L309K mutation offers a promising strategy to enhance the druggability properties of candidate antibodies.

An In Vitro Assay to Measure Antibody-Dependent Cellular Phagocytosis by Phagocytes

An In Vitro Assay to Measure Antibody-Dependent Cellular Phagocytosis by Phagocytes

An In Vitro Assay to Measure Antibody-Dependent Cellular Phagocytosis by Phagocytes

An In Vitro Assay to Measure Antibody-Dependent Cellular Phagocytosis by Phagocytes

Sequencing of anti-CD19 therapies in the management of diffuse large B-cell lymphoma.

Several second- and third-line immunotherapeutic options for patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) ineligible for autologous stem cell transplant are directed against the B-cell antigen CD19. The anti-CD19 monoclonal antibody tafasitamab, paired with the immunomodulator lenalidomide, mediates antibody-dependent cellular toxicity and cellular phagocytosis; the antibody-drug conjugate loncastuximab tesirine delivers the DNA-cross-linking agent tesirine via CD19 binding and internalization; and CD19-directed chimeric antigen receptor T-cell therapy (CAR-T) products are engineered from autologous T cells. While CD19 expression is assessed at diagnosis, clinically relevant thresholds of CD19 expression-which may not be detectable with current routine methodologies-have not been defined and may vary between CD19-directed treatment modalities. Determining optimal treatment sequencing strategies with CD19-directed therapy has been hampered by the exclusion of patients with prior CD19-directed therapies from major clinical trials. Antigen escape, attributed to mechanisms including epitope loss and defective cell-surface trafficking of CD19, is an important cause of CAR-T failure. Limited data suggest that CD19 expression may be maintained after non-CAR-T CD19-directed therapy and retrospective analyses indicate that some patients with disease that relapses after CAR-T may benefit from subsequent CD19-directed therapy. To date, clinical evidence on the effect of anti-CD19 therapy prior to CAR-T is restricted to small case series. Prospective studies and detailed analyses to understand how pre- and post-treatment CD19 expression correlates with clinical responses to subsequent CD19-directed therapy are needed to fully maximize treatment strategies.

Targeting CEACAM5-positive solid tumors using NILK-2401, a novel CEACAM5xCD47 κλ bispecific antibody.

Blocking the CD47 "don't eat me"-signal on tumor cells with monoclonal antibodies or fusion proteins has shown limited clinical activity in hematologic malignancies and solid tumors thus far. Main side effects are associated with non-tumor targeted binding to CD47 particularly on blood cells. We present here the generation and preclinical development of NILK-2401, a CEACAM5×CD47 bispecific antibody (BsAb) composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format). NILK-2401 is a fully human BsAb binding the CEACAM5 N-terminal domain on tumor cells by its lambda light chain arm with an affinity of ≈4 nM and CD47 with its kappa chain arm with an intendedly low affinity of ≈500 nM to enabling tumor-specific blockade of the CD47-SIRPα interaction. For increased activity, NILK-2401 features a functional IgG1 Fc-part. NILK-2401 eliminates CEACAM5-positive tumor cell lines (3/3 colorectal, 2/2 gastric, 2/2 lung) with EC50 for antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity ranging from 0.38 to 25.84 nM and 0.04 to 0.25 nM, respectively. NILK-2401 binds neither CD47-positive/CEACAM5-negative cell lines nor primary epithelial cells. No erythrophagocytosis or platelet activation is observed. Quantification of the pre-existing NILK-2401-reactive T-cell repertoire in the blood of 14 healthy donors with diverse HLA molecules shows a low immunogenic potential. In vivo, NILK-2401 significantly delayed tumor growth in a NOD-SCID colon cancer model and a syngeneic mouse model using human CD47/human SIRPα transgenic mice and prolonged survival. In cynomolgus monkeys, single doses of 0.5 and 20 mg/kg were well tolerated; PK linked to anti-CD47 and Fc-binding seemed to be more than dose-proportional for Cmax and AUC0-inf. Data were validated in human FcRn TG32 mice. Combination of a CEACAM5-targeting T-cell engager (NILK-2301) with NILK-2401 can either boost NILK-2301 activity (Emax) up to 2.5-fold or allows reaching equal NILK-2301 activity at >600-fold (LS174T) to >3,000-fold (MKN-45) lower doses. NILK-2401 combines promising preclinical activity with limited potential side effects due to the tumor-targeted blockade of CD47 and low immunogenicity and is planned to enter clinical testing.

Adaptive immune responses to two-dose COVID-19 vaccine series in healthy Canadian adults ≥ 50 years: a prospective, observational cohort study

To evaluate immune responses to COVID-19 vaccines in adults aged 50 years and older, spike protein (S)-specific antibody concentration, avidity, and function (via angiotensin-converting enzyme 2 (ACE2) inhibition surrogate neutralization and antibody dependent cellular phagocytosis (ADCP)), as well as S-specific T cells were quantified via activation induced marker (AIM) assay in response to two-dose series. Eighty-four adults were vaccinated with either: mRNA/mRNA (mRNA-1273 and/or BNT162b2); ChAdOx1-S/mRNA; or ChAdOx1-S/ChAdOx1-S. Anti-S IgG concentrations, ADCP scores and ACE2 inhibiting antibody concentrations were highest at one-month post-second dose and declined by four-months post-second dose for all groups. mRNA/mRNA and ChAdOx1-S/mRNA schedules had significantly higher antibody responses than ChAdOx1-S/ChAdOx1-S. CD8+ T-cell responses one-month post-second dose were associated with increased ACE2 surrogate neutralization. Antibody avidity (total relative avidity index) did not change between one-month and four-months post-second dose and did not significantly differ between groups by four-months post-second dose. In determining COVID-19 correlates of protection, a measure that considers both antibody concentration and avidity should be considered.

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.

The IL-7R antagonist lusvertikimab reduces leukemic burden in xenograft ALL via antibody-dependent cellular phagocytosis

AbstractAcute lymphoblastic leukemia (ALL) arises from the uncontrolled proliferation of B-cell precursors (BCP-ALL) or T cells (T-ALL). Current treatment protocols obtain high cure rates in children but are based on toxic polychemotherapy. Novel therapies are urgently needed, especially in relapsed/refractory (R/R) disease, high-risk (HR) leukemias and T-ALL, in which immunotherapy approaches remain scarce. Although the interleukin-7 receptor (IL-7R) plays a pivotal role in ALL development, no IL-7R–targeting immunotherapy has yet reached clinical application in ALL. The IL-7Rα chain (CD127)–targeting IgG4 antibody lusvertikimab (LUSV; formerly OSE-127) is a full antagonist of the IL-7R pathway, showing a good safety profile in healthy volunteers. Here, we show that ∼85% of ALL cases express surface CD127. We demonstrate significant in vivo efficacy of LUSV immunotherapy in a heterogeneous cohort of xenograft derived from patients (PDX) with BCP- and T-ALL in minimal residual disease (MRD) and overt leukemia models, including R/R and HR leukemias. Importantly, LUSV was particularly effective when combined with polychemotherapy in a phase 2-like PDX study with CD127high samples leading to MRD-negativity in >50% of mice treated with combination therapy. Mechanistically, LUSV targeted ALL cells via a dual mode of action comprising direct IL-7R antagonistic activity and induction of macrophage-mediated antibody-dependent cellular phagocytosis (ADCP). LUSV–mediated in vitro ADCP levels significantly correlated with CD127 expression levels and the reduction of leukemia burden upon treatment of PDX animals in vivo. Altogether, through its dual mode of action and good safety profile, LUSV may represent a novel immunotherapy option for any CD127+ ALL, particularly in combination with standard-of-care polychemotherapy.

Abstract 1365: INB03: a new immune checkpoint inhibitor that reprograms macrophage polarization, boosts ADCP and reverts T-cell exhaustion markers

Abstract Trastuzumab resistance is an important clinical issue in HER2+ breast cancer (BC), and few actionable targets are available. We demonstrated that soluble TNF (sTNF) upregulates mucin 4 (MUC4) expression, which shields trastuzumab epitope on HER2 hindering its therapeutic effect, and that sTNF blockade with INB03 decreases MUC4 expression which, together with trastuzumab, triggers an effective antitumor immune response based on antitumoral macrophage-NK cell collaboration. Because immune checkpoint molecules (ICP) and T cell exhaustion foster tumor immune escape, we addressed whether INB03 could modulate macrophage polarization, T cell exhaustion, and ICP expression in both populations to boost an antitumor immune response. For in vitro assays, human monocytes and T cells were isolated from buffy coats and enriched with specific RosetteSep. Macrophages (Mϕ) were differentiated for 6 days with M-CSF (10 ng/ml) to M0, and polarized for 48h to the M1-like subtype with LPS (50 ng/ml)+IFNγ (20 ng/ml), or to the M2-like subtype with IL-4 (20 ng/ml)+IL-10 (20 ng/ml). Then, INB03 was added (10 μg/ml) for 96h with corresponding cytokines to already-polarized Mϕ to test its ability to revert polarization. Also, Mϕ antibody-dependent cellular phagocytosis (ADCP) against human HER2+ breast cancer cell line JIMT-1 was assessed. T cells were activated with CD3/CD28 beads and cultured for 7 or 14 days. INB03 was added at those times for 48h to test its ability to modulate ICP and exhaustion markers. For in vivo assays, tumor-free mice or bearing the HER2+ C4HD tumor were treated with vehicle or INB03 (10 mg/kg, i.p.) for 21 days. Polarization, exhaustion and ICP markers were studied in Mϕ and T cells from spleen and tumor. Adding INB03 to already-polarized human Mϕ enhanced polarization towards M1-like (CD86+CD206−). Polarized Mϕ exposed to INB03 showed decreased PD1 and PDL-1 expression. Also, the expression of ADCP-inhibitory markers B7H4 and SIRPα was diminished when INB03 was present. These Mϕ exhibited increased ADCP against JIMT-1 cells treated with INB03, which showed downregulation of the inhibitory signal CD47. Murine Mϕ from tumor-bearing mice treated with INB03 showed increased polarization to the M1-like phenotype. Human CD8+ T cells showed decreased expression of PD1, PD-L1, LAG3, TIM3 and TIGIT. However, only TIM3 and PD1 were downregulated in murine CD8+ T cells of tumor-bearing mice treated with INB03. In all, sTNF blockade skews Mϕ to the M1-like phenotype and reprograms already-polarized pro-tumoral M2-like Mϕ to antitumoral ones. INB03 can tailor the tumor microenvironment by promoting Mϕ ADCP against tumor cells and by acting as an ICP inhibitor for CD8+ T cells, possibly relieving their exhaustion. HER2+MUC4+ BC patients could benefit from the administration of INB03 to boost targeted therapy efficacy and overcome tumor-induced macrophage and T-cell immune escape. Citation Format: Sofia Bruni, Maria F. Mercogliano, Roxana Schillaci. INB03: a new immune checkpoint inhibitor that reprograms macrophage polarization, boosts ADCP and reverts T-cell exhaustion markers [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 1365.

Abstract 1890: HMA800067, a novel CD38-targeting antibody-drug conjugate (ADC), demonstrated superior anti-tumor activity to daratumumab in preclinical B-cell malignancies models

Abstract Introduction: Daratumumab (Dara), an anti-CD38 monoclonal antibody, has been widely used in the treatment of multiple myeloma (MM). However, some of MM patients exhibit primary or acquired resistance to Dara therapy. A CD38 targeting antibody-drug conjugate (ADC) HMA800067 is developed, in which Dara was conjugated with cytotoxic payload via a novel linker, aiming to have superior anti-tumor efficacy to Dara, even in the subjects with resistance to Dara treatment. Methods: HMA800067 was characterized by ELISA-based binding assay, cell internalization assay, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) assays. The in vitro anti-tumor potency was assessed against a panel of 24 MM and B cell lymphoma cell lines with various CD38 expressions. Bystander effect of HMA800067 was evaluated in co-culture of CD38− SU-DHL-2 cells with CD38+ Ramos cells. In vivo anti-tumor efficacy was investigated in the multiple MM and Diffuse Large B-cell Lymphoma (DLBCL) subcutaneous xenograft models, including some non-responsive to Dara. Results: HMA800067 displayed comparable binding affinity and internalization capability to Dara in CD38+ Ramos cells. And HMA800067 maintained the ability of Dara to induce the death of CD38+ Daudi cells by ADCC and ADCP, which indicated that conjugation did not change the property of Dara in the ADC upon antigen binding. HMA800067 exhibited a CD38-dependent cytotoxic activity with median IC50 of 0.46 nM against a panel of tumor cell lines, with a trend that the higher CD38 expression, the stronger potency could be seen. Bystander killing effect of HMA800067 was observed in CD38− cells when co-cultured with CD38+ cells in vitro. Furthermore, with the low release of payload in plasma and sustainable high exposure of payload in the tumor, HMA800067 demonstrated dose-dependent and superior anti-tumor efficacy to Dara in all tested models, and 10 mg/kg induced complete tumor regression in MM RPMI-8226, DLBCL SU-DHL-6 and SU-DHL-10 models. For example, in SU-DHL-6 xenograft model, the treatment of intravenous injection of HMA800067 at 10 mg/kg single dose induced complete tumor regression in all the animals within 10 days of administration, which was maintained until the end of study (day 49), while 10 mg/kg Dara twice a week for 2 weeks inhibited tumor growth within 20%-40% without any tumor regression during a four-week observation post initial dosing. Conclusion: HMA800067 demonstrated potent anti-tumor activities in vitro and in vivo in multiple B-cell malignant tumor lines, including those with poor response to Dara. These results supported further development of HMA800067, as a superior therapeutic option for treatment of patients with CD38+ B cell malignancies. Citation Format: Yan Xu, Junqing Liang, Shuwen Jiang, Haibin Yang, Hui Zhang, Fangfang Mao, Jiahuan Zhu, Jianlin He, Na Yang, Jian Wang, WeiHan Zhang, Yongxin Ren, Weiguo Su. HMA800067, a novel CD38-targeting antibody-drug conjugate (ADC), demonstrated superior anti-tumor activity to daratumumab in preclinical B-cell malignancies models [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 1890.

Abstract 3716: Enhancing anti-tumor responses in triple-negative breast cancer by combining SIRPα inhibition with immunogenic chemotherapy or antibody-drug conjugates

Abstract Background: Triple-negative breast cancer (TNBC) is a subtype of breast cancer defined by the absence of hormone receptors and HER2 receptor on tumor cells. Because of a lack of targetable receptors and poor response to PD1/PDL1 immunotherapy, TNBC has limited systemic treatment options. In previous work using a genomic screen (Shuptrine et al., 2017), we identified CD47 as one of the leading factors that contributes to TNBC immune evasion. The CD47-SIRPα axis is an innate immune checkpoint that primarily regulates myeloid cells. In the context of cancer, tumor cells upregulate CD47 to engage SIRPα inhibitory signaling to block phagocytosis. While CD47 or SIRPα blockade alone show limited antitumor effect, therapies that combine blockade of this axis with other treatment agents, such as tumor-antigen specific antibodies, show superior tumor inhibition. Thus, we hypothesize that the addition of SIRPα inhibition to existing TNBC treatments (chemotherapy and Trop2-antibody drug conjugates (ADC)) will improve their treatment efficacy. Methods: To ascertain the impact of SIRPα inhibition combined with chemotherapy and Trop2-ADC, we utilized and validated a novel SIRPα KO mouse, along with SIRPα mAbs, in our previously utilized models of Triple-Negative Breast Cancer (TNBC) (Crosby et al., 2018). Using both in vitro and in vivo tumor models, we tested the therapeutic impact of SIRPα inhibition/blockade combined with different immunogenic chemotherapies (doxorubicin/cyclophosphamide) and Sacituzumab govitecan (SG) in TNBC. Results: After validating the loss of SIRPα in KO mice, we found that E0771 TNBC cells had comparable growth rates in WT and SIRPα KO mice. Similarly, we did not observe different anti-tumor responses to Doxorubicin between WT and SIRPα-KO mice. However, we observed more profound suppression of E0771 tumors after cyclophosphamide treatment in SIRPα KO mice than in WT mice. To evaluate the efficacy of SG in inducing antibody-dependent cellular phagocytosis (ADCP), we co-cultured SIRPα KO and WT mouse macrophages with a syngeneic mouse TNBC model (MM3MG-Trop2-WT). We found that SG promotes ADCP in WT macrophages, and SIRPα loss further increased this ADCP, suggesting the potential importance of antibody-mediated phagocytosis as a mechanism of action for SG. In vivo, we implanted human TNBC cells (MDA-MB-468) in mice and treated them with SG with and without SIRPα antibody blockade. These studies demonstrated an anti-tumor effect from SG that was potentially enhanced by SIRPα blockade, suggesting the clinical potential of this combination in TNBC. Conclusion: Our study demonstrates that the addition of SIRPα inhibition to chemotherapy or SG may promote tumor phagocytosis and stimulate stronger antitumor immunity in TNBC. These results support further investigation of SIRPα-targeting combination therapies for TNBC. Citation Format: Xingru Ma, Li-Chung Tsao, Tao Wang, Cong-Xiao Liu, Melissa Gajda, Xiao Yang, Gangjun Lei, Junping Wei, Zachary Hartman. Enhancing anti-tumor responses in triple-negative breast cancer by combining SIRPα inhibition with immunogenic chemotherapy or antibody-drug conjugates [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 3716.

Abstract 2722: CTN001, a first Twin Fc-immune cell engager, with a superior potency against Her2+ and Her2-low cancers by engaging multiple Fc-mediated mechanisms

Abstract Many therapeutic monoclonal antibodies mediate the immune effector functions through the engagement of their fragment crystallizable (Fc) domain and Fcγ receptors on the immune cells. However, these functions are limited due to the low affinity of their interactions and the requirement of abundancy of antigens for the avidity effects. Recently, mutation-based Fc engineered antibodies showed their enhanced immune effector functions mostly through the NK cell-mediated ADCC. To overcome the limited improvement of current Fc engineering, we developed an innovated platform, Twin Fc-Immune Cell Engager (Twin Fc-ICE) composed of a single Fab domain and two Fc domain with drug-like properties. Here we demonstrated the superior efficacy of CTN001, a first Twin Fc-ICE, in in vitro and in vivo Her2+ tumor models. CTN001 bound to various types of Fcγ receptor-expressing immune cells with stronger affinity, supported by its better in vitro affinity to all types of Fcγ receptors than wild type and Fc-engineered antibodies. To evaluate the pharmacological efficacies, CTN001 was tested for its functions of in vitro antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) and in vivo efficacy with Her2+ xenograft models. In Her2+ models, both CTN001 and trastuzumab induced ADCC-mediated effects, but CTN001 showed better anti-tumor cytotoxicity through ADCP and CDC than trastuzumab. Importantly, CTN001 also showed cytotoxic effects on Her2-low tumor cells. Consistently, CTN001 showed better anti-tumor efficacy than trastuzumab or its combination with pertuzumab in Her2+ tumor xenograft model and, surprisingly, even in Her2-low or resistant models. These results suggest that CTN001 can provide new therapeutic options for treating Her2-low patients differentiated from antibody-drug conjugates (ADCs), and that the Twin Fc-ICE can be an innovative platform to overcome limitations of existing therapeutics. Citation Format: Deokjae Lee, Sujin Yoon, Jiseon Bae, Minju Kim, Ki-June Lee, Eunmi Kim, Hyunkyu Park, Eun S. Choi, Gi-Hyeok Yang. CTN001, a first Twin Fc-immune cell engager, with a superior potency against Her2+ and Her2-low cancers by engaging multiple Fc-mediated mechanisms [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 2722.

Abstract 2739: XON27, a novel and potent immunotherapy against hematologic malignancies

Abstract Background: Over the past decades, intensive research has led to the development of innovative therapies (monoclonal and bispecific antibodies, CAR T cells) that have increased the survival rate of patients with multiple myeloma (MM) and B cell lymphoma. However, despite this progress, the majority of patients relapse and become refractory due to the development of resistance. Polyclonal antibodies, with their ability to target multiple antigens, can counteract this resistance. Here we evaluate the safety and efficacy of XON27, a first-in-class glyco-humanized polyclonal antibody (GH-pAb), in MM and other blood cancers. Material and Methods: The ability of XON27 to induce antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) and apoptosis was tested in a panel of hematological malignancy cells and peripheral blood mononuclear cells (PBMC). Specific binding to human blood cancers was assessed using tissue microarrays. To study resistance mechanisms, MM cell lines were cocultured with macrophages in the presence or absence of XON27 and macrophage. To study relapse mechanisms and tumorigenicity, repeated in vitro administration of XON27 was evaluated in KMS-12-BM. The in vivo efficacy of XON27 was evaluated in KMS-12-BM NMRI nude mice model. Pharmacokinetics and safety of this drug were assessed in non-human primate after repeated IV dosing up to 60mg/kg.Results: XON27 induces potent and specific cytotoxicity in several blood malignancy cell lines including human MM cells via multiple mechanisms: CDC (EC50=35.84 ug/ml), apoptosis (IC50=100 ug/ml) and ADCP (up to 40% of cell death at 50 ug/ml). The cytotoxicity induced by XON27 in the KMS-12-BM was significantly more potent compared to daratumumab (anti-CD38) in both CDC and apoptosis assays, XON27 was able to kill up to 100% of cancer cells without affecting PBMC. XON27 showed cross-reactivity with blood cancer patient biopsies without staining healthy tissue. In addition, repeated administrations of XON27 demonstrate its ability to effectively kill tumor cells after each administration. No resistance is induced after repeated treatments. Interestingly, XON27 blocked the proliferation of residual tumor cells for up to 45 days after the second treatment at 50 ug/ml compared to the control condition, demonstrating sustainable effect of XON27 on tumorigenicity. The in vitro efficacy of XON27 was translated into in vivo efficacy in a murine MM xenograft model. It induced a significant reduction in tumor growth of up to 75%. XON27 was also characterized by a high tolerability profile and satisfactory exposure in primates. Conclusion: Although many therapeutic advances have been made in the field of blood cancers, myeloma remains an incurable disease with a real need for new innovative therapies. XON27 represents a promising and selective immunotherapy which may provide an effective and safe treatment against refractory myeloma. Citation Format: Carine Ciron, Pierre Morice, Françoise Shneiker, Odile Duvaux, Firas Bassissi. XON27, a novel and potent immunotherapy against hematologic malignancies [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 2739.

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

Abstract Background: Several approaches such as antibody drug conjugate (ADC), chimeric antigen receptor T cells (CAR-T) and recently bispecific antibodies are being introduced as innovative weapon in B-Cell lymphoma treatment. In the opposite, very limited therapeutic options are available for the treatment of rare refractory T cell malignancies such as PTCL. The 5-year survival rate of PTCL is lower than 30% according to the recent clinical data. Developing new effective treatment strategies for PTCL is an urgent problem. LIS22, is a first in class glyco-humanized polyclonal antibody (GH-pAb), targeting multiple tumor-associated antigens simultaneously. In this study, we characterized the safety and efficacy of LIS22 in preclinical models of T cell malignancies. Material and Methods: LIS22 ability to induce Antibody-Dependent Cell Cytotoxicity (ADCC), Antibody-Dependent Cellular Phagocytosis (ADCP), Complement Dependent Cytotoxicity (CDC), and apoptosis was tested in a panel of hematologic malignancy cell lines and peripheral blood mononuclear cell (PBMC). To assess the targeting and recognition of LIS22 in PTCL patients, we evaluated the immunolabelling of LIS22 on patients’ biopsies (n=119) using tissue microarray. LIS22 efficacy in vivo were evaluated in NMRI nude mice and SRG rats using T1301 and Jurkat cancer cell lines. Pharmacokinetics and safety of this drug were assessed in cynomolgus monkeys after single and repeated IV dosing up to 50mg/kg. Results: LIS22 acts via several mechanisms, at 30µg/ml, it induced cytotoxicity via CDC (in 70%), ADCP (in 49%), ADCC (in 41%) and apoptosis (in 30%) of HPB-ALL human T blood cancer cell line but not in PBMC. LIS22 showed a potent in vitro antitumor activity in a panel of hematologic malignancy cell lines, it induced specific tumor cell CDC (EC50=41.4±28.9µg/mL). In both CDC and apoptosis cytotoxicity assays, LIS22 displayed a significantly higher potency on T cell blood cancers and no toxicity on healthy blood cells compared to PTCL clinically active drug Alemtuzumab (Anti-CD52). It was able to kill up to 100% of the cancer cells without affecting PBMC. In immunolabeling assay, LIS22 demonstrated cross-reaction to PTCL patient biopsy (staining up to 93%). In vivo efficacy studies in different mice and rat xenograft models, LIS22 induced a significant reduction of tumor growth up to 90% across several tested tumors. LIS22 was also characterized by high tolerance profile and satisfactory exposure in monkeys after repeated dosing (up to 250mg/kg of cumulative dose). Conclusion: LIS22 appeared as a novel and promising cancer immunotherapy against T Cell hematologic malignancies. It has already been administered to human in another indication and showed a satisfactory PK and safety profile (no signs of immunotoxicity or systemic cytokine release). LIS22 is now ready to enter Phase I/II in the coming months for the treatment of peripheral T cell lymphoma (PTCL). Citation Format: Carine Ciron, Francoise Shneiker, Ophélie Dauphouy, Juliette Rousse, Pierre-Joseph Royer, Gwenaelle Evanno, Odile Duvaux, Firas Bassissi. LIS22, a first in class polyclonal antibody immunotherapy in T cell blood cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2737.

Abstract 2836: Assessment of therapeutic antibody efficacy without the interference of murine Fc receptors allows for investigation of human antibody-dependent cellular cytotoxicity mediated by NK cells in the FcResolv™ hIL-15 NOG mouse model

Abstract Targeted antibody therapy is applied to treat various cancer types. In addition to the primary mode of action (MOA), which involves direct binding to the tumor antigen, indirect MOA acting through the constant region (Fc) of the antibody can enhance anti-tumor efficacy. Indirect mechanisms engage the innate immune system, mediated by both the complement system (complement-dependent cytotoxicity (CDC)) and immune cells (antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC)). These indirect mechanisms can complicate the evaluation and accurate assessment of antibody-induced ADCC by human NK cells in current mouse models. In immune-deficient mouse strains (e.g. NOG), false positives and/or negatives may occur due to interactions with murine Fc receptors. These can either result in anti-tumor responses via activation of the murine innate immune system or can interfere with the human-targeted therapy's primary MOA. To study the response to anti-cancer antibodies without the interference of these murine Fc receptor interactions and to investigate ADCC mediated by human NK cells, a novel mouse model deficient in Fc receptors and expressing human IL-15 (FcResolv™ hIL-15 NOG) was employed for testing antibody therapies. Methods: Patient-derived xenograft (PDX) tumor models were transplanted into hIL-15 NOG and FcResolv™ hIL-15 NOG mice. A human head and neck squamous cell carcinoma and a lung adenocarcinoma PDX model were both treated with cetuximab. Treatment with pertuzumab and trastuzumab was applied in a breast ductal carcinoma PDX model. Rituximab treatment was tested in two diffuse large B cell lymphoma PDX models. Based on growth kinetics, the lung cancer PDX model was chosen for further testing of ADCC in the NK cell-humanized FcResolv™ hIL-15 NOG mouse. Results and Conclusion: There was no difference in percent tumor growth inhibition between the FcResolv™ hIL-15 NOG and hIL-15 NOG mice with regards to cetuximab treatment in the lung and head and neck cancer or for trastuzumab treatment of breast ductal carcinoma. However, pertuzumab treatment revealed a false positive efficacy, with the false positive effect more pronounced in hIL-15 NOG mice than in FcResolv™ hIL-15 NOG mice. In one of the lymphoma models, a false negative result was observed. Here, rituximab did not show notable tumor inhibition in the hIL-15 NOG mice but did in the FcResolv™ hIL-15 NOG mice. These results demonstrate that FcResolv™ hIL-15 NOG mice serve as a suitable mouse model for a more accurate assessment of the therapeutic efficacy of anti-tumor antibodies. Additionally, evaluation of human-mediated ADCC of therapeutic antibodies in NK cell-humanized FcResolv™ hIL-15 NOG allows detection of effects specifically mediated by human NK cells. Citation Format: Simone Rhein, Maria Stecklum, Monika Buczek, Janell Richardson, Jens Hoffmann. Assessment of therapeutic antibody efficacy without the interference of murine Fc receptors allows for investigation of human antibody-dependent cellular cytotoxicity mediated by NK cells in the FcResolv™ hIL-15 NOG mouse model [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 2836.

Abstract 88: Novel cell-based bioassays enable preclinical animal studies during immuno-oncology drug development

Abstract Translation of immuno-oncology research into novel therapies relies on animal models to advise target selection and portray mechanism of action (MoA) and pharmacokinetics of lead candidates. Mouse, monkey, and more recently, canine models of human cancers are being utilized to this end. However, one major challenge in developing and utilizing these models is the lack of technology to characterize non-human analogs of therapeutic antibodies. Herein, we report the development of novel cell-based bioassays for mouse, monkey, and canine targets to enable immuno-oncology preclinical studies. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are important MoAs for antibody-based cancer immunotherapies. We have developed a suite of cell-based bioassays, each expressing a corresponding animal Fcγ receptor (FcγR) and a luciferase reporter that responds to FcγR activation. These assays exhibit sensitive and quantitative luciferase readouts of FcγR activation. Animal models are further used to provide insight into the potential efficacy of immune checkpoint (IC) inhibitors. Programmed cell death protein 1 (PD-1) is an IC receptor that negatively regulates T cell function. Blockade of PD-1 has demonstrated clinical efficacy, but there remains a significant fraction of patients unresponsive to this therapy. Combination of PD-1 inhibitors with other interventions has been a significant focus for many programs. However, tools for functional validation of non-human PD-1 inhibitors are lacking. We report here the development of two new bioassays for functional characterization of PD-1/PD-L1 inhibitors for mouse and canine targets. Each of these assays consists of two engineered cell lines: a T effector cell line that express a luciferase reporter driven by specific promoter/response elements responding to the intracellular signals mediated by the T cell receptor (TCR), with modulation from PD-1, and an artificial antigen presenting cell line (aAPC). The bioassays are sensitive, quantitative, and have demonstrated characteristics required for potency assays for validation of antibodies in preclinical combination studies. Citation Format: Jun Wang, Denise Garvin, Aileen Paguio, Elizabeth Perrin, Jim Hartnett, Mei Cong, Jamison Grailer. Novel cell-based bioassays enable preclinical animal studies during immuno-oncology drug development [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 88.

Abstract 5244: SIRPα knockout iPSC-derived macrophages (iMACs) are resistant to CD47-dependent inhibition of phagocytosis and efficiently kill tumor cells in pre-clinical models

Abstract The CD47-SIRPα axis is a critical checkpoint that prevents SIRPα-positive macrophages from phagocytosing CD47-expressing solid tumors. Several agents aiming to block this axis have recently entered early clinical trials including anti-CD47 and anti-SIRPα monoclonal antibodies (mAb). These checkpoint inhibitors (CPI) aim to modulate the phagocytotic activity of endogenous tumor associated macrophages (TAMs). However, the adoptive transfer of macrophages resistant to CD47-based inhibition in the tumor microenvironment (TME) could also increase clinical efficacy while avoiding side effects linked to the use of anti-CD47 mAb. Cell therapy based on autologous macrophages have gained increasing attention for cancer treatment due to their ability to infiltrate into the immunosuppressive TME and their unique immunomodulatory characteristics. However, due to required intricate genetic manipulation of autologous macrophage cell product for each patient, optimization and consistency of the cell product remains challenging. In contrast, the use of induced pluripotent stem cell (iPSC)-derived macrophages (iMACs) facilitates the introduction of genetic modifications to further optimize the iMAC cell product and limits the need for combination therapies. The knockout (KO) of the SIRPα gene in iMACs is a promising genetic modification that results in a potent iMAC cell therapy product resistant to phagocytosis inhibition by CD47-expressing tumor cells. A SIRPα KO was introduced in a fully characterized GMP iPSC line that was then differentiated to iMACs using Evotec’s 3D differentiation protocol. The SIRPα KO iMACs were then evaluated for their antibody-dependent cellular phagocytosis (ADCP) capacity in comparison to antibody-loaded wildtype (WT) iMACs when co-cultured with CD47-expressing tumor cells. SIRPα KO iPSCs showed differentiation potential comparable to WT iPSC and resulted in iMACs expressing typical markers of fully differentiated macrophages. SIRPα KO iMACs exhibited increased phagocytic potency and killing capacity compared to WT iMACs when both cell types were exposed to CD47-positive tumor cells and loaded with the same tumor-targeting mAb. This increased phagocytosis of tumor cells by antibody-loaded SIRPα KO iMACs was also comparable to ADCP observed for WT iMACs in the presence of an anti-CD47 blocking antibody. Using Evotec’s gene editing platform we were able to efficiently generate SIRPα-deficient iPSCs that serve as the starting material to manufacture highly pure, genetically modified iMACs that lack SIRPα expression rendering them resistant to CD47-dependent inhibition of phagocytosis. This novel allogeneic off-the-shelf iMAC cell product overcomes the need to combine this cell therapeutic with CD47-SIRPα axis CPIs and provides the basis to develop innovative treatments for solid tumors. Citation Format: Kathrin Haake, Michela Mirenda, Quentin Bernard, Philip Hublitz, Lucie Gouxette, Martin Briscadieu, Philine Scheinpflug, Garima Singh, Alica Hinkelmann, Tanja Schneider, Michael Esquerré, Audrey Holtzinger, Michael Epstein, Daniel Sommermeyer, Michael Paillasse, Andreas Scheel, Markus Dangl, Monika Braun, Nadja Wagner. SIRPα knockout iPSC-derived macrophages (iMACs) are resistant to CD47-dependent inhibition of phagocytosis and efficiently kill tumor cells in pre-clinical models [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 5244.

Abstract 2693: MicroRNA manipulation of macrophage polarization in DLBCL to augment antibody immunotherapy

Abstract Background: CD163 positive tumor associated macrophages (TAM), have been associated with poor response to immunochemotherapy in DLBCL. Targeting these TAMs to reverse immunosuppression is one approach for improving treatment response rates. MicroRNAs have the potential to modify hundreds of genes by interacting with multiple mRNA targets, thereby altering the global transcriptional profile of cells. Manipulation of microRNAs may prove an effective means of switching the transcriptomic profiles of TAMs to more activated, anti-tumor states resulting in the reversal of immune resistance and enhancement of treatment efficacy. Methods: MicroRNAs differentially expressed in DLBCL and associated with poor response were identified using whole gene correlated network analysis of DLBCL datasets. Peripheral blood mononuclear cells (PBMCs) were obtained from normal blood donors and differentiated into macrophages following cell culture with MCSF. Macrophages were polarized using immunosuppressive (IL4 + IL13) or immunostimulating (LPS + IFNγ) cytokines and microRNA expression measured using RT-qPCR. To assess antibody dependent cellular phagocytosis (ADCP), macrophages were generated and polarized as above and transfected with microRNA inhibitors (Thermofisher) prior to co-incubation with target cells. Human Chronic Lymphocytic Leukemia (CLL) cells were labelled with CSFE and used as target cells with Rituximab as the ADCP-inducing monoclonal antibody (mAb) and Herceptin as an isotype control. The percentage of Fc gamma receptor IIIA (FcγRIIIA)-expressing macrophages taking up target cells was determined using flow cytometry and analyzed using Flowjo software. Diagnostic biopsies from patients were then analyzed for microRNA 142-5p and CD206 expression using miRNAScope assays and immunohistochemistry. Results: There was significant upregulation of microRNA 142-5p in macrophages polarized with IL4 plus IL13 compared to macrophages polarized with LPS plus IFNγ and non-polarized macrophages. Inhibition of microRNA 142-5p in IL4 plus IL13 polarized macrophages led to a significant increase in FcγRIIIA, CD40 and CD47 expression resulting in enhanced phagocytosis of CLL cells opsonized with Rituximab plus anti-CD47 mAb when compared with controls. High microRNA 142-5p expression in combination with high CD206 expression predicted poor response to immunochemotherapy in 38 treatment naïve human DLBCL samples (median PFS 12 months v NR p=0.0096). Discussion: Transfection of a microRNA 142-5p inhibitor can repolarize immunosuppressive macrophages to a more anti-tumor phenotype leading to enhanced ADCP. CD206 positive TAM infiltration in combination with high microRNA 142-5p expression predicted poor outcome following treatment with immunochemotherapy in DLBCL. It may be possible to use microRNA directed therapies targeting the tumor microenvironment to enhance treatment efficacy. Citation Format: Jemma Longley, Russell Foxall, Steven Thirdborough, Steve Beers, Mark Cragg. MicroRNA manipulation of macrophage polarization in DLBCL to augment antibody immunotherapy [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 2693.

Abstract 2059: An image-based real time method to measure antibody-dependent cellular phagocytosis

Abstract Antibody-dependent cellular phagocytosis (ADCP) is an important mechanism of action of therapeutic antibodies. In vitro quantification of macrophage phagocytosis has been technically challenging. Flow cytometry assays were traditionally used for ADCP activity measurements, these methods were static measurements at certain time point, thus provide no information about engulfment kinetics. Moreover, the flow cytometry assays require lifting the macrophage cells from the labware which may affect macrophage activities. We have developed a real time ADCP assay using a live cell analysis system. The target cells labeled with a pH sensitive dye only fluoresce when they are engulfed in acidic phagosomes in the presence of specific antibodies. Our assay is a plate-based with a simple mix and read protocol without disturbing the macrophages. The new method is quantitative and robust, can be readily used for high-throughput screening for functional antibodies. By studying the ADCP activities of different subtypes of macrophages, we found M2 microphages showed the strongest ADCP activity compare to M0 and M1 macrophages, due to their different expression levels of FcgRs. The assay is highly sensitive to differentiate the antibody variants with enhanced Fc function. It can also be used to study the kinetics and mechanisms of ADCP. Citation Format: Jianyong Wang, Yongchang Shi, Yonglian Sun, James T. Koerber, Akiko Seki, Sascha Rutz. An image-based real time method to measure antibody-dependent cellular phagocytosis [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 2059.