Antibody-dependent Cellular Phagocytosis Research Articles

Dual Fc optimization to increase the cytotoxic activity of a CD19-targeting antibody.

Targeting CD19 represents a promising strategy for the therapy of B-cell malignancies. Although non-engineered CD19 antibodies are poorly effective in mediating complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), these effector functions can be enhanced by Fc-engineering. Here, we engineered a CD19 antibody with the aim to improve effector cell-mediated killing and CDC activity by exchanging selected amino acid residues in the Fc domain. Based on the clinically approved Fc-optimized antibody tafasitamab, which triggers enhanced ADCC and ADCP due to two amino acid exchanges in the Fc domain (S239D/I332E), we additionally added the E345K amino acid exchange to favor antibody hexamerization on the target cell surface resulting in improved CDC. The dual engineered CD19-DEK antibody bound CD19 and Fcγ receptors with similar characteristics as the parental CD19-DE antibody. Both antibodies were similarly efficient in mediating ADCC and ADCP but only the dual optimized antibody was able to trigger complement deposition on target cells and effective CDC. Our data provide evidence that from a technical perspective selected Fc-enhancing mutations can be combined (S239D/I332E and E345K) allowing the enhancement of ADCC, ADCP and CDC with isolated effector populations. Interestingly, under more physiological conditions when the complement system and FcR-positive effector cells are available as effector source, strong complement deposition negatively impacts FcR engagement. Both effector functions were simultaneously active only at selected antibody concentrations. Dual Fc-optimized antibodies may represent a strategy to further improve CD19-directed cancer immunotherapy. In general, our results can help in guiding optimal antibody engineering strategies to optimize antibodies’ effector functions.

Myeloid checkpoint blockade improves killing of T-acute lymphoblastic leukemia cells by an IgA2 variant of daratumumab.

Antibody-based immunotherapy is increasingly employed to treat acute lymphoblastic leukemia (ALL) patients. Many T-ALL cells express CD38 on their surface, which can be targeted by the CD38 antibody daratumumab (DARA), approved for the treatment of multiple myeloma. Tumor cell killing by myeloid cells is relevant for the efficacy of many therapeutic antibodies and can be more efficacious with human IgA than with IgG antibodies. This is demonstrated here by investigating antibody-dependent cellular phagocytosis (ADCP) by macrophages and antibody-dependent cell-mediated cytotoxicity (ADCC) by polymorphonuclear (PMN) cells using DARA (human IgG1) and an IgA2 isotype switch variant (DARA-IgA2) against T-ALL cell lines and primary patient-derived tumor cells. ADCP and ADCC are negatively regulated by interactions between CD47 on tumor cells and signal regulatory protein alpha (SIRPα) on effector cells. In order to investigate the impact of this myeloid checkpoint on T-ALL cell killing, CD47 and glutaminyl-peptide cyclotransferase like (QPCTL) knock-out T-ALL cells were employed. QPTCL is an enzymatic posttranslational modifier of CD47 activity, which can be targeted by small molecule inhibitors. Additionally, we used an IgG2σ variant of the CD47 blocking antibody magrolimab, which is in advanced clinical development. Moreover, treatment of T-ALL cells with all-trans retinoic acid (ATRA) increased CD38 expression leading to further enhanced ADCP and ADCC, particularly when DARA-IgA2 was applied. These studies demonstrate that myeloid checkpoint blockade in combination with IgA2 variants of CD38 antibodies deserves further evaluation for T-ALL immunotherapy.

Characterization of Plasma Immunoglobulin G Responses in Elite Neutralizers of Human Cytomegalovirus.

Human cytomegalovirus (HCMV) is the most common infectious complication of organ transplantation and cause of birth defects worldwide. There are limited therapeutic options and no licensed vaccine to prevent HCMV infection or disease. To inform development of HCMV antibody-based interventions, a previous study identified individuals with potent and broad plasma HCMV-neutralizing activity, termed elite neutralizers (ENs), from a cohort of HCMV-seropositive (SP) blood donors. However, the specificities and functions of plasma antibodies associated with EN status remained undefined. We sought to determine the plasma antibody specificities, breadth, and Fc-mediated antibody effector functions associated with the most potent HCMV-neutralizing responses in plasma from ENs (n = 25) relative to that from SP donors (n = 19). We measured antibody binding against various HCMV strains and glycoprotein targets and evaluated Fc-mediated effector functions, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). We demonstrate that ENs have elevated immunoglobulin G binding responses against multiple viral glycoproteins, relative to SP donors. Our study also revealed potent HCMV-specific antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis activity of plasma from ENs. We conclude that antibody responses against multiple glycoprotein specificities may be needed to achieve potent plasma neutralization and that potently HCMV elite-neutralizing plasma antibodies can also mediate polyfunctional responses.

Maternal Fc-mediated non-neutralizing antibody responses correlate with protection against congenital human cytomegalovirus infection.

Human cytomegalovirus (HCMV) is the most common congenital infection and a leading cause of stillbirth, neurodevelopmental impairment, and pediatric hearing loss worldwide. Development of a maternal vaccine or therapeutic to prevent congenital HCMV has been hindered by limited knowledge of the immune responses that protect against HCMV transmission in utero. To identify protective antibody responses, we measured HCMV-specific IgG binding and antiviral functions in paired maternal and cord blood sera from HCMV-seropositive transmitting (n = 41) and non-transmitting (n = 40) mother-infant dyads identified via a large, US-based, public cord blood bank. We found that high-avidity IgG binding to HCMV and antibody-dependent cellular phagocytosis (ADCP) were associated with reduced risk of congenital HCMV infection. We also determined that HCMV-specific IgG activation of FcγRI and FcγRII was enhanced in non-transmitting dyads and that increased ADCP responses were mediated through both FcγRI and FcγRIIA expressed on human monocytes. These findings suggest that engagement of FcγRI/FcγRIIA and Fc effector functions including ADCP may protect against congenital HCMV infection. Taken together, these data can guide future prospective studies on immune correlates against congenital HCMV transmission and inform HCMV vaccine and immunotherapeutic development.

Impact of adjuvants on the biophysical and functional characteristics of HIV vaccine-elicited antibodies in humans

Adjuvants can alter the magnitude, characteristics, and persistence of the humoral response to protein vaccination. HIV vaccination might benefit from tailored adjuvant choice as raising a durable and protective response to vaccination has been exceptionally challenging. Analysis of trials of partially effective HIV vaccines have identified features of the immune response that correlate with decreased risk, including high titers of V1V2-binding IgG and IgG3 responses with low titers of V1V2-binding IgA responses and enhanced Fc effector functions, notably antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). However, there has been limited opportunity to compare the effect of different adjuvants on these activities in humans. Here, samples from the AVEG015 study, a phase 1 trial in which participants (n = 112) were immunized with gp120SF-2 and one of six different adjuvants or combinations thereof were assessed for antibody titer, biophysical features, and diverse effector functions. Three adjuvants, MF59 + MTP-PE, SAF/2, and SAF/2 + MDP, increased the peak magnitude and durability of antigen-specific IgG3, IgA, FcγR-binding responses and ADCP activity, as compared to alum. While multiple adjuvants increased the titer of IgG, IgG3, and IgA responses, none consistently altered the balance of IgG to IgA or IgG3 to IgA. Linear regression analysis identified biophysical features including gp120-specific IgG and FcγR-binding responses that could predict functional activity, and network analysis identified coordinated aspects of the humoral response. These analyses reveal the ability of adjuvants to drive the character and function of the humoral response despite limitations of small sample size and immune variability in this human clinical trial.

ICAM-1-binding Plasmodium falciparum erythrocyte membrane protein 1 variants elicits opsonic-phagocytosis IgG responses in Beninese children

Members of the highly polymorphic Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on the surface of infected erythrocytes (IEs) are important virulence factors, which mediate vascular adhesion of IEs via endothelial host receptors and are targets of naturally acquired immunity. The PfEMP1 family can be divided into clinically relevant subgroups, of which some bind intercellular adhesion molecule 1 (ICAM-1). While the acquisition of IgG specific for ICAM-1-binding DBLβ domains is known to differ between PfEMP1 groups, its ability to induce antibody-dependent cellular phagocytosis (ADCP) is unclear. We therefore measured plasma levels of DBLβ-specific IgG, the ability of such IgG to inhibit PfEMP1-binding to ICAM-1, and its ability to opsonize IEs for ADCP, using plasma from Beninese children with severe (SM) or uncomplicated malaria (UM). IgG specific for DBLβ from group A and B ICAM-1-binding PfEMP1 were dominated by IgG1 and IgG3, and were similar in SM and UM. However, levels of plasma IgG inhibiting ICAM-1-binding of group A DBLβ of PFD1235w was significantly higher in children with UM than SM, and acute UM plasma induced a higher ADCP response than acute SM plasma.

Multiepitope supramolecular peptide nanofibers eliciting coordinated humoral and cellular antitumor immune responses.

Subunit vaccines inducing antibodies against tumor-specific antigens have yet to be clinically successful. Here, we use a supramolecular α-helical peptide nanofiber approach to design epitope-specific vaccines raising simultaneous B cell, CD8+ T cell, and CD4+ T cell responses against combinations of selected epitopes and show that the concurrent induction of these responses generates strong antitumor effects in mice, with significant improvements over antibody or CD8+ T cell–based vaccines alone, in both prophylactic and therapeutic subcutaneous melanoma models. Nanofiber vaccine–induced antibodies mediated in vitro tumoricidal antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). The addition of immune checkpoint and phagocytosis checkpoint blockade antibodies further improved the therapeutic effect of the nanofiber vaccines against murine melanoma. These findings highlight the potential clinical benefit of vaccine-induced antibody responses for tumor treatments, provided that they are accompanied by simultaneous CD8+ and CD4+ responses, and they illustrate a multiepitope cancer vaccine design approach using supramolecular nanomaterials.

A homogeneous bioluminescent immunoassay for parallel characterization of binding between a panel of antibodies and a family of Fcγ receptors

Fc engineering efforts are increasingly being employed to modulate interaction of antibodies with variety of Fc receptors in an effort to improve the efficacy and safety of the therapeutic antibodies. Among the various Fc receptors, Fc gamma receptors (FcγRs) present on variety of immune cells are especially relevant since they can activate multiple effector functions including antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP). Depending on the desired mechanism of action (MOA) of the antibody, interactions between Fc domain of the antibody and FcγR (denoted as Fc/FcγR) may need to be enhanced or abolished. Therefore, during the antibody discovery process, biochemical methods are routinely used to measure the affinities of Fc/FcγR interactions. To enable such screening, we developed a plate based, simple to use, homogeneous immunoassays for six FcγRs by leveraging a luminescent protein complementation technology (NanoBiT). An added advantage of the NanoBiT immunoassays is their solution-based format, which minimizes well known surface related artifacts associated with traditional biosensor platforms (e.g., surface plasmon resonance and biolayer interferometry). With NanoBiT FcγRs assays, we demonstrate that assays are specific, report IgG subclass specific affinities and detect modulation in Fc/FcγR interactions in response to the changes in the Fc domain. We subsequently screen a panel of therapeutic antibodies including seven monoclonal antibodies (mAbs) and four polyclonal intravenous immunoglobulin (IVIg) products and highlight the advantages of parallel screening method for developing new antibody therapies.

Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities.

Soluble angiotensin-converting enzyme 2 (ACE2) constitutes an attractive antiviral capable of targeting a wide range of coronaviruses using ACE2 as their receptor. Using structure-guided approaches, we developed a series of bivalent ACE2-Fcs harboring functionally and structurally validated mutations that enhance severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain recognition by up to ~12-fold and remove angiotensin enzymatic activity. The lead variant M81 potently cross-neutralized SARS-CoV-2 variants of concern (VOCs), including Omicron, at subnanomolar half-maximal inhibitory concentration and was capable of robust Fc-effector functions, including antibody-dependent cellular cytotoxicity, phagocytosis, and complement deposition. When tested in a stringent K18-hACE2 mouse model, Fc-enhanced ACE2-Fc delayed death by 3 to 5 days or effectively resolved lethal SARS-CoV-2 infection in both prophylactic and therapeutic settings via the combined effects of neutralization and Fc-effector functions. These data add to the demonstrated utility of soluble ACE2 as a valuable SARS-CoV-2 antiviral and indicate that Fc-effector functions may constitute an important component of ACE2-Fc therapeutic activity.

Venetoclax enhances the efficacy of therapeutic antibodies in B-cell malignancies by augmenting tumor cell phagocytosis

AbstractImmunotherapy has evolved as a powerful tool for the treatment of B-cell malignancies, and patient outcomes have improved by combining therapeutic antibodies with conventional chemotherapy. Overexpression of antiapoptotic B-cell lymphoma 2 (Bcl-2) is associated with a poor prognosis, and increased levels have been described in patients with “double-hit” diffuse large B-cell lymphoma, a subgroup of Burkitt's lymphoma, and patients with pediatric acute lymphoblastic leukemia harboring a t(17;19) translocation. Here, we show that the addition of venetoclax (VEN), a specific Bcl-2 inhibitor, potently enhanced the efficacy of the therapeutic anti-CD20 antibody rituximab, anti-CD38 daratumumab, and anti-CD19-DE, a proprietary version of tafasitamab. This was because of an increase in antibody-dependent cellular phagocytosis by macrophages as shown in vitro and in vivo in cell lines and patient-derived xenograft models. Mechanistically, double-hit lymphoma cells subjected to VEN triggered phagocytosis in an apoptosis-independent manner. Our study identifies the combination of VEN and therapeutic antibodies as a promising novel strategy for the treatment of B-cell malignancies.

Maternal immune response and placental antibody transfer after COVID-19 vaccination across trimester and platforms

The availability of three COVID-19 vaccines in the United States provides an unprecedented opportunity to examine how vaccine platforms and timing of vaccination in pregnancy impact maternal and neonatal immunity. Here, we characterize the antibody profile after Ad26.COV2.S, mRNA-1273 or BNT162b2 vaccination in 158 pregnant individuals and evaluate transplacental antibody transfer by profiling maternal and umbilical cord blood in 175 maternal-neonatal dyads. These analyses reveal lower vaccine-induced functions and Fc receptor-binding after Ad26.COV2.S compared to mRNA vaccination and subtle advantages in titer and function with mRNA-1273 versus BN162b2. mRNA vaccines have higher titers and functions against SARS-CoV-2 variants of concern. First and third trimester vaccination results in enhanced maternal antibody-dependent NK-cell activation, cellular and neutrophil phagocytosis, and complement deposition relative to second trimester. Higher transplacental transfer ratios following first and second trimester vaccination may reflect placental compensation for waning maternal titers. These results provide novel insight into the impact of platform and trimester of vaccination on maternal humoral immune response and transplacental antibody transfer.

Penpulimab, an Fc-Engineered IgG1 Anti-PD-1 Antibody, With Improved Efficacy and Low Incidence of Immune-Related Adverse Events.

BackgroundIgG4 anbibodies are deficient in stability and may contribute to tumor-associated escape from immune surveillance. We developed an IgG1 backbone anti-programmed cell death protein-1 (PD-1) antibody, penpulimab, which is designed to remove crystallizable fragment (Fc) gamma receptor (FcγR) binding that mediates antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and proinflammatory cytokine release.MethodsAggregation of different anti-PD-1 antibodies was tested by size exclusion chromatography, and melting temperature midpoint (Tm) and aggregation temperature onset (Tagg) were also determined. The affinity constants of penpulimab for PD-1 and human FcγRs were measured by surface plasmon resonance and biolayer interferometry. ADCC and ADCP were determined in cellular assays and antibody-dependent cytokine release (ADCR) from human macrophages was detected by ELISA. Binding kinetics of penpulimab to human PD-1 was determined by Biacore, and epitope/paratope mapping of PD-1/penpulimab was investigated using x-ray crystallography. Additionally, patients from six ongoing trials were included for analysis of immune-related adverse events (irAEs).ResultsPenpulimab demonstrated better stability and a lower level of host-cell protein residue compared with IgG4 backbone anti-PD-1 antibodies. As expected, penpulimab exhibited no apparent binding to FcγRIa, FcγRIIa_H131, FcγRIIIa_V158 and FcγRIIIa_F158, elicited no apparent ADCC and ADCP activities, and induced no remarkable IL-6 and IL-8 release by activated macrophages in vitro. Penpulimab was shown in the co-crystal study to bind to human PD-1 N-glycosylation site at N58 and had a slower off-rate from PD-1 versus nivolumab or pembrolizumab. Four hundred sixty-five patients were analyzed for irAEs. Fifteen (3.2%) patients had grade 3 or above irAEs. No death from irAEs was reported.ConclusionsIgG1 backbone anti-PD1 antibody penpulimab has a good stability and reduced host cell protein residue, as well as potent binding to the antigen. Fc engineering has eliminated Fc-mediated effector functions of penpulimab including ADCC, ADCP and reduced ADCR, which may contribute to its more favorable safety profile.Clinical Trial Registration www.ClinicalTrials.gov, identifier: AK105-101: NCT03352531, AK105-201: NCT03722147, AK105-301: NCT03866980, AK105-202:NCT03866967, AK105-203: NCT04172571, AK105-204: NCT04172506.

P1249: INHIBITION OF THE PENTOSE-PHOSPHATE-PATHWAY (PPP) MEDIATES MACROPHAGE REPROGRAMMING FROM SUPPORTIVE BYSTANDER FUNCTION TOWARDS ANTIBODY-DEPENDENT LYMPHOMA CELL CLEARANCE AND LYMPHOMA SUPPRESSION

Background: Treatment response in lymphoma depends on the interaction and functional status of macrophages as important effector cells for tumor cell clearance by antibody-dependent cellular phagocytosis (ADCP). In the tumor microenvironment (TME) there is a limited supply of nutrients which implicates metabolic reprograming of immune cells such as changed phagocytic capacity of macrophages. Aims: We aimed to identify metabolic pathways regulating macrophages in the TME to improve macrophage effector cell function in lymphoma therapy. Methods: We addressed macrophage lymphoma cell co-cultures using macrophage cell lines, primary murine macrophages and human monocyte-derived macrophages. For pathway inhibition specific compounds as well as shRNA-mediated knockdown were used for functional read-out of ADCP and viability in lymphoma co-culture. Macrophage reprogramming was assessed by microscopy, FACS-immunophenotyping, ELISA, (phospho)-proteomic and metabolomic assessment. C57BL/6 mice and the humanized aggressive lymphoma model hMB were used for in vivo treatment with the pentose phosphate pathway (PPP) inhibitor S3 and analysis of macrophage programming and overall survival. Results: Inhibition of AMP kinase, glycolysis or mitochondrial ATP-production did not increase the phagocytosis rate in ADCP co-cultures. Inhibiting the PPP induced an increased lymphoma cell phagocytosis by macrophages. Under PPP-inhibition, the oxygen consumption and the glycolysis of macrophages was increased and the cells developed a more activated morphology with formation of filopodia, which are needed for phagocytosis initiation. Moreover, a down-regulation of immune-inhibitory surface proteins PD-L1 and SIRP1α was seen and cytokine secretion was shifted from anti-inflammatory IL10 to pro-inflammatory IL6 secretion. PPP-inhibition of macrophages significantly reduced CLL-cell support in vitro abrogating the “nurse-like” effect of macrophages. The increased lymphoma cell clearance and phenotypic alterations of macrophages were also observed in PPP-enzyme knockdown of transketolase (TKT) and 6-phosphogluconatedehydrogenase (6PGD) in macrophages and by inhibiting the PPP in primary murine and human macrophages. In a multiomics assessment of PPP-inhibition on proteome, phosphoproteome and metabolome level, we found protein expression alterations regulating metabolism and immunity. A connection between PPP inhibition and changed immune profile by modulation of the Stat1-Irg1-itaconate axis was identified and validated. In a humanized lymphoma mouse model the addition of PPP-inhibitor S3 led to significant prolonged survival of lymphoma bearing mice and an increased maturation and pro-inflammatory polarization of macrophages. Summary/Conclusion: An increased maturation, activation and repolarization of macrophages towards a pro-inflammatory and phagocytic active phenotype was observed by inhibiting the pentose-phosphate-pathway (PPP). This led to a significant survival benefit in in vivo lymphoma mouse model. We were able to point out the underlying Stat1-Irg1-itaconate signalling axis, connecting metabolic regulation and immune-phenotype of macrophages. We hypothesize the PPP as a key regulator of macrophage function determining the support of malignant B cell growth versus the capacity of lymphoma cell clearance and thereby therapy outcome. By that, we have identified the PPP as a targetable modulator of macrophage polarization to improve efficacy of B cell lymphoma and CLL therapy.

Abstract 1766: SGN-ALPV a novel, investigational vedotin ADC demonstrates highly effective targeting of oncofetal phosphatases ALPP and ALPPL2 in preclinical models

Abstract Antibody-drug conjugates (ADC) leverage the specificity of antibodies to direct the delivery of potent cytotoxic agents to cancer cells. To fully leverage this specificity, targets with significant differential tumor versus normal tissue expression are ideal for ADC development. Placental alkaline phosphatases, ALPP and ALPPL2, are proteins present during fetal development but are found in several tumor types, making them an attractive ADC target. These cell membrane-attached phosphatases form homo- and heterodimers and play key roles in nucleotide recycling. SGN-ALPV is a novel investigational vedotin ADC comprised of a humanized IgG1 monoclonal antibody conjugated to the microtubule disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable peptide linker that has been clinically validated in multiple ADC programs. Here we characterize the target antigens, ALPP and ALPPL2, and evaluate SGN-ALPV antitumor activity in preclinical models. Immunohistochemistry and genomic data mining showed that ALPP and ALPPL2 have a highly restricted normal tissue expression yet high expression in several solid tumor types including ovarian, endometrial, germ cell, non-small cell lung and gastric carcinomas. Importantly, normal tissue expression is restricted to placenta and reproductive tissue, with low levels in lung tissue. SGN-ALPV utilizes a humanized antibody, h12F3, that is highly specific for both human and cynomolgus monkey ALPP and ALPPL2 proteins, but not other related phosphatases. In vitro, upon binding to SGN APLV, the antigens, ALPP and ALPPL2, are internalized to lysosomal vesicles releasing the MMAE payload, whose cytotoxic features drive mitotic arrest, apoptosis, and the induction of immunogenic cell death. Additionally, SGN-ALPV mediates antibody dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in vitro but lacks complement-dependent cytotoxicity. In preclinical studies, SGN-ALPV exhibits robust antitumor activity in cell line- and patient-derived xenograft models of ovarian, lung, pancreatic, and gastric carcinoma including models with both homogenous and heterogeneous expression of ALPP and ALPPL2, consistent with robust bystander activity of vedotin ADCs. Although, the contribution of ADCC and ADCP in vivo is currently unknown, it is expected that the antitumor activity of SGN-ALPV is mediated by MMAE cytotoxicity. SGN-ALPV was well tolerated in non-human primates (NHP) and exhibited linear pharmacokinetic characteristics, with a toxicity profile consistent with other vedotin-based ADCs. In summary, differential expression of ALPP and ALPPL2 in the tumor versus normal tissue, antibody specificity, antitumor activity, and tolerability of SGN-ALPV provide a strong rationale for the initiation of a planned first-in-human Phase 1 clinical study. Citation Format: Sarah Anderson, Nanna Hansen, Robert Lawrence, Aroon T. Chande, David Ortiz, Christopher Carosino, Esther Trueblood, Nicole Stevens, Kerry Klussman, Angela Epp, Bill Arthur, Shyra Gardai, Hector Rincon. SGN-ALPV a novel, investigational vedotin ADC demonstrates highly effective targeting of oncofetal phosphatases ALPP and ALPPL2 in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1766.

Abstract LB179: Patient-derived, vaccine-elicited, anti-MUC1 antibodies directly target tumor cells for elimination via multiple immune mechanisms

Abstract During premalignant and malignant transformation, cells undergo antigenic changes that can be recognized by the immune system, and this can be leveraged both for immune prevention and therapy. One such change occurs with MUC1, or Mucin-1, a large variable number tandem repeat (VNTR)-containing transmembrane protein that is overexpressed and hypoglycosylated on a majority of precancerous and adenocarcinoma cells, including those of the colon, lung, breast, pancreas and ovaries. This tumor form of MUC1 is recognized by both cellular and humoral immunity. Healthy individuals at high risk of developing colon cancer due to a history of colonic polyps were enrolled in a clinical trial testing the feasibility of prophylactic vaccination against the tumor form of MUC1, also expressed on polyps (Kimura et al., 2013). Half the participants generated high levels of anti-MUC1 IgG antibodies, some of which were cloned to further analyze their tumor specificity and to potentially develop them as therapeutics for patients with cancer (Lohmueller et al., 2016). As these fully human IgG1 monoclonal antibodies (mAbs) were selected and affinity matured in healthy individuals who experienced no negative side effects to the vaccine in >10 years, it increases the likelihood that as therapeutics they will be safe. To study anti-tumor efficacy and mechanism(s) of action of 12 of these mAbs, tumor cells were co-incubated with immune cells in the presence of the mAbs, and each mAb’s ability to mediate antibody-dependent cellular cytotoxicity (ADCC) by NK cells, antibody-dependent cellular phagocytosis (ADCP) by monocytes, antibody-dependent trogocytosis/trogoptosis (ADCT) by neutrophils, antibody-dependent cytokine release (ADCR) and complement dependent cytotoxicity (CDC) was assessed. Several MUC1 mAbs examined could mediate ADCC, ADCP, ADCT and ADCR, with some mechanisms being cell-line dependent. One mAb was selected for in vivo testing and found to delay breast cancer growth and prolong survival in immunocompromised mice lacking T cells, B cells and NK cells, pointing to ADCP and ADCT as important anti-tumor functions. Additionally, the relationship between epitope location and immune effector functions was explored by creating different MUC1 constructs that vary epitope distance from the cell surface, number of epitopes, etc, to test properties of the target antigen that may affect antibody efficacy. These latter experiments revealed an increased efficiency in phagocytosis and trogocytosis when the mAbs bound an epitope that was both proximal to and anchored to the membrane. Notably, unlike published studies of rituximab and alemtuzumab, the amount of MUC1 mAb binding did not always correlate with increased effector function. Our results may inform not only the development of these MUC1-based therapies but more broadly future therapeutic mAb design against a variety of targets. Citation Format: Michelle L. McKeague, Jason Lohmueller, Matthew T. Dracz, Najla Saadallah, Jia Xue, Eric D. Ricci, William Lu, Olivera J. Finn. Patient-derived, vaccine-elicited, anti-MUC1 antibodies directly target tumor cells for elimination via multiple immune mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB179.

Abstract 6008: Effective depletion of tumor-infiltrating Tregs by a novel anti-CCR8 antibody (LM-108): Addressing resistance associated with immune checkpoint inhibitors

Abstract Immune checkpoint inhibitors (ICIs) have generated durable clinical responses in multiple tumor types, but their therapeutic efficacy is limited by development of primary or acquired resistance. The tumor microenvironment (TME) contains many factors influencing ICI responses. The ratio of effector T-cells (Teffs) and regulatory T-cells (Tregs) in tumors has been shown to be associated with the efficacy of ICIs in murine models, indicating that increasing Teffs or decreasing Tregs in tumor may prevent development of resistance to immunotherapy. There is a high and restricted expression of CC motif chemokine receptor 8 (CCR8) on tumor-infiltrating Tregs, whereas they are negligibly expressed on Teffs. Deletion of tumor-infiltrating Tregs via murine anti-CCR8 surrogates has shown effective anti-tumor activity in tumor models.LM-108 is a novel humanized anti-CCR8 monoclonal antibody with engineered IgG1 that enhances antibody-dependent cell-mediated cytotoxicity (ADCC). The expression of CCR8 and Foxp3 on tumor-infiltrating lymphocytes (TILs) was analyzed using published single-cell sequencing data. The binding affinity of LM-108 was assessed by flow cytometry using CCR8 high- and low-expressing cell lines. In vitro measurements of ADCC and antibody-dependent cellular phagocytosis (ADCP) mediated by LM-108 were conducted in CCR8 overexpressing HEK293, CHO-K1, and Jurkat cells. Furthermore, in vivo anti-tumor activity of LM-108 as monotherapy and in combination with anti-PD-1 therapy was evaluated in syngeneic models and PD-1-resistant tumor models with human CCR8 knock-in mice. Analysis of published single-cell sequencing data showed that in comparison with CCR4, CCR8 gene expression was specifically overlapped with Foxp3 in TILs of breast cancer. Fluorescence-activated cell sorting (FACS) assay revealed that LM-108 specifically binds with high affinity to human CCR8 in a dose-dependent manner. ADCC and ADCP effects induced by LM-108 were observed in HEK293 and CHO-K1 cells with different levels of CCR8 expression. In vivo data of CT26 syngeneic model showed that LM-108 murine surrogate antibody alone or in combination with anti-PD-1 antibody significantly inhibited tumor growth. Treatment with LM-108 effectively depleted tumor-infiltrating Tregs accompanied by increase in CD8/Treg ratio in tumors, leading to significant anti-tumor activity in MC38 model. Finally, LM-108 showed a synergistic therapeutic efficacy in PD-1 resistant models in combination with anti-PD-1 antibody. To conclude, LM-108 is a novel Fc-optimized CCR8 antibody that selectively depletes tumor infiltrating Tregs thereby improving anti-tumor immune response either as monotherapy or combination therapy. Thus, LM-108 can be a promising therapeutic approach to overcome ICI resistance in cancer patients. Citation Format: Jie Luo, Wentao Huang, Junwei Yang, Jin Li, Yifan Li, Da Fei, Xia Qin, Runsheng Li. Effective depletion of tumor-infiltrating Tregs by a novel anti-CCR8 antibody (LM-108): Addressing resistance associated with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6008.

Abstract 3430: CD47 x ICAM-1 bispecific antibody represents a novel approach for treating ICAM-1 overexpressing tumors

Abstract CD47/SIRPa axis is an important checkpoint of innate immune system, CD47 interacts with its ligand signal regulatory protein-alpha (SIRPa) on myeloid cells, conveys a “don’t eat me” signal and blocks macrophage mediated phagocytosis. Tumor cells, which express high level of CD47, exploit this mechanism to evade from immune surveillance. CD47 is considered a prominent target for cancer treatment. However, the wide expression of CD47 on normal cells could cause antigen sink and lead to safety issues, such as anemia and thrombocytopenia. ICAM-1 is constitutively present at low levels on endothelial cells, but its overexpression has been observed on various types of tumors, such as primary myeloma, lymphoma and kidney cancer. Here we describe a novel CD47 x ICAM-1 bispecific antibody, which specifically targets CD47+/ICAM-1+ tumors in preclinical studies.A novel anti-CD47 antibody and an anti-ICAM-1 antibody were used for the generation of a CD47 x ICAM-1 bispecific antibody. In vitro assays including FACS binding, FACS-based SIRPa blocking, ADCP, RBC binding and hemagglutination were performed to characterize the bispecific CD47 x ICAM-1 antibody. In vivo efficacies of CD47 x ICAM-1 bispecific antibody were evaluated in xenograft tumor models with high ICAM-1 expression.The CD47 x ICAM-1 bispecific antibody selectively exhibited potent SIRPa blocking and antibody-dependent cellular phagocytosis (ADCP) activity on CD47+/ICAM-1+ tumor cells, but not on cells lacking ICAM-1 expression. Minimal RBC binding and RBC phagocytosis were observed upon treatment with CD47 x ICAM-1 bispecific antibody. The bispecific antibody did not cause any appreciable hemagglutination with up to 1µM antibody treatment. Most importantly, the CD47 x ICAM-1 bispecific antibody demonstrated potent anti-tumor activities in in vivo CDX and PDX models that overexpress both CD47 and ICAM-1. Our findings suggest that the novel CD47 x ICAM-1 bispecific antibody selectively binds to CD47 and blocks CD47/SIRPa binding on ICAM1 overexpressing tumor cells. The bispecific antibody has minimal RBC binding compared to the bivalent CD47 monoclonal antibodies and benchmark anti-CD47 antibodies. The bispecific antibody shows potent in vivo efficacy and specificity toward ICAM-1+/CD47+ positive tumor cells and represents a novel approach for treating ICAM-1+/CD47+ tumors. Citation Format: Xinhua Wang, Oi Kwan Wong, Leonard Post, Xiaocheng Chen. CD47 x ICAM-1 bispecific antibody represents a novel approach for treating ICAM-1 overexpressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3430.

Abstract 2866: BAY 3375968: An afucosylated anti-CCR8 antibody depleting activated intratumoral regulatory T cells as a cancer immunotherapy

Abstract Regulatory T cells (Tregs) play an indispensable role in mediating peripheral tolerance to self-antigens. They can also promote tumor growth by suppressing the function of effector CD8+ and CD4+ T cells in the tumor microenvironment (TME). Furthermore, Tregs are identified as one of the key resistance mechanisms hampering the efficacy of immune checkpoint inhibitors (ICIs) across many tumor types. Therefore, there is a high need for safe and effective agents that would specifically deplete tumor-infiltrating Tregs while sparing both peripheral Tregs and effector T cells. Chemokine receptor 8 (CCR8) is predominantly expressed on activated tumor-infiltrating Tregs and marks the most suppressive and proliferative Treg population. CCR8+ Tregs are associated with high tumor grade and poor overall survival across many tumor types such as lung, breast, or head-neck cancer. Thus, unlike other approaches directed against Tregs, targeting CCR8 offers the opportunity to specifically deplete intra-tumoral Tregs without impacting peripheral Tregs or other immune cells. BAY 3375968 is a non-internalizing fully human glycoengineered (afucosylated) monoclonal IgG1 antibody, which in vitro selectively depleted human CCR8+ Tregs via antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP). In vivo efficacy studies using mouse surrogate antibodies showed strong monotherapeutic efficacy across a variety of murine tumor models with clear correlation of intratumoral CCR8+ Treg depletion and CD8+ T cell increase. The monotherapeutic efficacy of CCR8 depleting antibodies was further enhanced by combinations with ICIs. BAY 3375968 also showed a good safety profile in cynomolgus monkeys. In conclusion, CCR8 is a novel Treg depleting immunotherapy target, and due to its highly tumor-restricted expression profile, BAY 3375968 may provide superior clinical safety and efficacy profile comparing to other less specific Treg targeting approaches. Based on the promising pre-clinical data, preparations for a phase I clinical trial investigating the safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary anti-tumor activity of BAY 3375968 are underway. Citation Format: Helge Roider, Su-Yi Tseng, Sabine Hoff, Sandra Berndt, Katharina Filarski, Uwe Gritzan, Beatrix Stelte-Ludwig, Wiebke M. Nadler, Joanna Grudzinska-Goebel, Philipp Ellinger, Mark Trautwein, Matyas Gorjanacz. BAY 3375968: An afucosylated anti-CCR8 antibody depleting activated intratumoral regulatory T cells as a cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2866.

Abstract 2120: In vitro suppressive bioassays using macrophages for the evaluation of immuno-oncology drug

Abstract Targeted activation of tumor infiltrating lymphocytes in the tumor microenvironment (TME) with so-called immune checkpoint inhibitors (ICIs) has resulted in the development of revolutionizing therapeutic anti-tumor strategies. A deepening understanding of the TME has followed in the ability to see beyond current immune checkpoint strategies. It becomes clear that players like Tumor Associated Macrophages (TAM) and Myeloid Derived Suppressor Cells (MDSC) play a significant role by downregulating anti-tumor responses. Their presence and roles in the TME open novel possibilities for modulation of the TME. To study these mechanisms, bioassays mimicking the suppressive activity of these cells on lymphocytes were developed. With regulatory functions in both innate and adaptive immune responses and different phenotypic profiles that classically are divided into M1-like and M2-like, macrophages undeniably represent important players in the TME. Where classically activated M1-like macrophages comprise immune effector cells with an inflammatory phenotype, alternatively activated M2-like macrophages display suppressive activities. Correspondingly, a variety of solid tumors are found to be enriched with these M2-like macrophages, suppressing anti-tumor immunity. The diverse roles of macrophage subtypes are still being explored and their phenotypic classification is likely more complex than the classical M1-like and M2-like. However, in vitro assays based on these 2 subtypes can be a first step to screen for the impact of test molecules on the phenotype and function of macrophages and their subsequent effect on lymphocytes. Using in vitro polarization and functional assays, the impact of test molecules on M1-like and M2-like macrophage generation and polarization can be assessed. The impact of test molecules on macrophage functionality can be further evaluated using a macrophage suppressive assay. Here the ability of test molecules to enhance the stimulating effect of M1-like macrophages or to reverse the suppressive effect of M2-like macrophages on lymphocytes can be evaluated by assessing their proliferation and cytokine production. Moreover, the potential stimulatory effect of test compounds on macrophages to perform Antibody-dependent cellular phagocytosis (ADCP) of tumor cells in co-culture assays could be another strategy to review therapeutic potential. Collectively, the development of novel bioassays contributes to a better understanding of the TME and thereby illuminates the steps required to elicit anti-tumor immune responses. It further aids the functional assessment of potential of new drugs, the design of clinical trials and the discovery of relevant biomarkers. Citation Format: Thibaut J. Janss, Simon Lefevre, Martijn Vlaming, Johan Arnold, Ellen Boelen, Sofie Pattijn. In vitro suppressive bioassays using macrophages for the evaluation of immuno-oncology drug [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2120.

Abstract 1281: SGN-B7H4V shows immunomodulatory activity through induction of immunogenic cell death

Abstract SGN-B7H4V is a novel investigational antibody-drug conjugate composed of a B7-H4-directed human monoclonal antibody conjugated to the validated vedotin drug linker, which incorporates the microtubule disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable peptide linkage. This vedotin drug linker system has been clinically validated by multiple ADC programs, including brentuximab vedotin, enfortumab vedotin, tisotumab vedotin, and polatuzumab vedotin. B7-H4 is an immune checkpoint ligand with elevated expression on a variety of solid tumor types, including breast, ovarian, and endometrial tumors. SGN-B7H4V is designed to bind and internalize the B7-H4/ADC complex from the surface of malignant cells and release the cytotoxic payload MMAE. The antitumor activity of SGN-B7H4V may be multimodal as SGN-B7H4V can induce tumor cell death through several mechanisms, including MMAE-mediated direct cytotoxicity and bystander killing as well as antibody-mediated functions including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Previously, vedotin ADCs have been described to elicit antitumor immune responses in part through induction of immunogenic cell death (ICD) mediated by the MMAE payload. These immunomodulatory effects potentially position vedotin ADCs to uniquely synergize with checkpoint inhibitors, supported by recent clinical activity observed when vedotin ADCs are paired with anti-PD1 agents. Here, we characterize SGN-B7H4V-mediated ICD and subsequent immunomodulatory activity. We also evaluate the contribution of SGN-B7H4V-induced immune activation to antitumor activity in combination with an anti-PD1 agent in an immunocompetent mouse model. In vitro, tumor cells treated with SGN-B7H4V showed several hallmarks of immunogenic cell death, including calreticulin exposure and release of ATP. In vivo, treatment of B7-H4-expressing tumors with SGN-B7H4V led to immune changes in the tumor microenvironment, including recruitment of macrophages and T cells. Finally, SGN-B7H4V drove robust, curative activity in an immunocompetent tumor model as a monotherapy and paired well with an anti-PD1 agent. Altogether, these data support the evaluation of SGN-B7H4V as a monotherapy in a first-in-human phase 1 clinical study and potential future clinical combinations with immunotherapies. Citation Format: Elizabeth E. Gray, Michelle Ulrich, Angela Epp, Patrick Younan, Kelly Hensley, Sean Allred, Julie Hahn, Kristen Gahnberg, Piper M. Treuting, John J. Gosink, Robert Thurman, Alyson J. Smith, Jason Schrum, Natalya Nazarenko, Shyra J. Gardai. SGN-B7H4V shows immunomodulatory activity through induction of immunogenic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1281.