Antibody-dependent Cell-mediated Phagocytosis Research Articles

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.

Rhamnose modified antibodies show improved immune killing towards EGFR-positive solid tumor cells

Therapeutic monoclonal antibodies (mAbs) against the epidermal growth factor receptor (EGFR) have shown clinical efficacy in colorectal cancer and other solid cancers. Enhancing the effector functions of these anti-EGFR mAbs is believed to be a valuable approach to achieve improved efficacy in clinical setting. Here, we report the development of an effector function-enhanced antibody by rhamnose (Rha) functionalization. Cetuximab, a human/mouse chimeric anti-EGFR mAb, was selected and site-specifically conjugated with Rha haptens. The obtained cetuximab-Rha conjugate was shown to be able to selectively redirect amounts of endogenous anti-Rha antibodies onto EGFR-positive solid tumor cells and thereby provide more Fc domains to achieve enhancement of effector functions including complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated phagocytosis (ADCP). Particularly, CDC, one powerful cell killing mechanism which is inactive in cetuximab, was dramatically improved. This study demonstrates the potential of rhamnose-modified antibody for EGFR-positive solid tumor immunotherapy.

New Frontiers in Monoclonal Antibodies for Relapsed/Refractory Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive lymphoma. Approximately 60% of patients are cured with R-CHOP as a frontline treatment, while the remaining patients experience primary refractory or relapsed disease (R/R). The prognosis for R/R DLBCL patients who are neither eligible for autologous stem-cell transplantations nor CAR-T-cell treatment is poor, representing an important unmet need. Monoclonal antibodies (mAbs) have dramatically improved therapeutic options in anti-cancer strategies, offering new opportunities to overcome chemo-refractoriness in this challenging disease, even in cases of primary non-responder DLBCL. Several novel mAbs, characterized by different mechanisms of action and targets, are now available for R/R DLBCL. Unbound mAbs induce an immune response against cancer cells, triggering different mechanisms, including antibody-dependent cellular cytotoxicity (ADCC), activation of antibody-dependent cell-mediated phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). Antibody-drug conjugates (ADCs) and radioimmunotherapy (RIT), respectively, deliver a cytotoxic payload or a beta-emitter radionuclide to the targeted cells and nearby bystanders. Bispecific T-cell engagers (BiTes) and immune checkpoint inhibitors (ICIs) redirect and enhance the immune response against tumor cells. Here, we review therapeutic strategies based on monoclonal antibodies for R/R DLBCL.

A Novel Fc-Optimized Antibody Drug Conjugate Targeting CD7 As a Therapeutic Strategy in T-Cell Acute Lymphoblastic Leukemia

Despite progress in improving treatment regimens for patients with T-cell acute lymphoblastic leukemia (T-ALL), the therapeutic options are still limited, and especially antibody-based immunotherapy is not established. The CD7 antigen represents a promising target structure in T-ALL since it is strongly expressed in different T-ALL subtypes including early T-cell precursor (ETP)-ALL. Therefore, different approaches are currently pursued for targeting CD7, including CAR-T cell therapy. Due to its high internalization capacity CD7 also represents an ideal target structure for antibody drug conjugates (ADC). Here, a novel antibody engineering approach for CD7-targeting was evaluated in vitro and in xenograft mouse models of T-ALL. A CD7 antibody was optimized for its ability to trigger antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) by introducing two amino acid substitutions (S239D/I332E; DE-variant) in the Fc-domain. In addition, the Fc-engineered antibody was conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) via an enzymatic-cleavable linker (mc-vc-PABC). The Fc-optimized ADC, CD7-DE-vcMMAE, as well as the unconjugated antibody, CD7-DE, showed improved binding to activating Fcγ receptors (FcγRIIa, FcγRIIIa) compared to a CD7 antibody lacking the DE-modification. This resulted in potent ADCC- and ADCP-activity against different T-ALL cell lines mediated by NK-cells or macrophages as effector cells (EC 50 ADCC, 1-7 pM). In contrast to the CD7-DE antibody, CD7-DE-vcMMAE showed strong cytotoxic effects independently of immune effector cell engagement by releasing its cytotoxic compound into T-ALL cells in an antigen-restricted manner, thereby inducing G2/M cell cycle arrest and apoptosis. CD7-DE-vcMMAE was active at subnanomolar concentrations demonstrating dose-dependent cytotoxic effects in six T-ALL cell lines (IC 50 0.2 - 1 nM). The extent of maximum growth inhibition ranged between 54-98 % and correlated with CD7 antigen density. Yet, although CD7-DE-vcMMAE did not kill CD7-negative cells directly, the linker design facilitated bystander killing activity. Thus, in co-culture experiments using CEM cells and CEM-CD7-knockout cells mimicking CD7-antigen escape, the ADC demonstrated significant killing of neighbouring CD7-negative cells, thereby extending its mode of action. The antitumor activity of the CD7-ADC was further investigated in xenograft mouse models of T-ALL. In a first model, CEM cells were injected subcutaneously into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and CD7-DE-vcMMAE treatment was evaluated in comparison to the unconjugated antibody CD7-DE. Treatment with CD7-DE-vcMMAE led to a significantly reduced tumor growth in comparison to CD7-DE or untreated animals (p<0.05; see panel A). A preclinical phase II-like patient-derived xenograft (PDX) study employing eight randomly selected T-ALL-PDX samples from pediatric and adult patients was conducted. PDX-cells were injected intravenously into NSG mice and treatment was started when the leukemia load reached 1 % human blasts in the peripheral blood, reflecting an overt leukemia situation. Animals receiving therapy with CD7-DE-vcMMAE showed significant prolongation of median survival in comparison to animals treated with a similarly designed control ADC targeting an irrelevant antigen (control-DE-vcMMAE) or which were left untreated (median survival time; CD7-DE-vc-MMAE: 82 days; untreated/control-DE-vcMMAE: 60 days; p<0.05; see panel B). Importantly, no leukemic blasts were found in the peripheral blood, spleen or bone marrow in animals treated with CD7-DE-vcMMAE and surviving the experimental period of 150 days. Together, the novel ADC CD7-DE-vcMMAE showed a unique set of Fc effector functions, potent direct growth inhibitory effects, bystander killing activity and efficacy in xenograft models of T-ALL. These results exhibit CD7-DE-vcMMAE as a promising therapeutic strategy and form the basis for new approaches in the treatment of T-ALL.

A Fully Human IgE Specific for CD38 as a Potential Therapy for Multiple Myeloma.

Multiple myeloma (MM) is an incurable malignancy of plasma cells and the second most common hematologic malignancy in the United States. Although antibodies in clinical cancer therapy are generally of the IgG class, antibodies of the IgE class have attractive properties as cancer therapeutics, such as their high affinity for Fc receptors (FcεRs), the low serum levels of endogenous IgE allowing for less competition for FcR occupancy, and the lack of inhibitory FcRs. Importantly, the FcεRs are expressed on immune cells that elicit antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and/or antigen presentation such as mast cells, eosinophils, macrophages, and dendritic cells. We now report the development of a fully human IgE targeting human CD38 as a potential MM therapy. We targeted CD38 given its high and uniform expression on MM cells. The novel anti-CD38 IgE, expressed in mammalian cells, is properly assembled and secreted, exhibits the correct molecular weight, binds antigen and the high affinity FcεRI, and induces degranulation of FcεRI expressing cells in vitro and also in vivo in transgenic BALB/c mice expressing human FcεRIα. Moreover, the anti-CD38 IgE induces ADCC and ADCP mediated by monocytes/macrophages against human MM cells (MM.1S). Importantly, the anti-CD38 IgE also prolongs survival in a preclinical disseminated xenograft mouse model using SCID-Beige mice and human MM.1S cells when administered with human peripheral blood mononuclear cells (PBMCs) as a source of monocyte effector cells. Our results suggest that anti-CD38 IgE may be effective in humans bearing MM and other malignancies expressing CD38.

Activity of tafasitamab in combination with rituximab in subtypes of aggressive lymphoma.

Despite recent advances in the treatment of aggressive lymphomas, a significant fraction of patients still succumbs to their disease. Thus, novel therapies are urgently needed. As the anti-CD20 antibody rituximab and the CD19-targeting antibody tafasitamab share distinct modes of actions, we investigated if dual-targeting of aggressive lymphoma B-cells by combining rituximab and tafasitamab might increase cytotoxic effects. Antibody single and combination efficacy was determined investigating different modes of action including direct cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in in vitro and in vivo models of aggressive B-cell lymphoma comprising diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL). Three different sensitivity profiles to antibody monotherapy or combination treatment were observed in in vitro models: while 1/11 cell lines was primarily sensitive to tafasitamab and 2/11 to rituximab, the combination resulted in enhanced cell death in 8/11 cell lines in at least one mode of action. Treatment with either antibody or the combination resulted in decreased expression of the oncogenic transcription factor MYC and inhibition of AKT signaling, which mirrored the cell line-specific sensitivities to direct cytotoxicity. At last, the combination resulted in a synergistic survival benefit in a PBMC-humanized Ramos NOD/SCID mouse model. This study demonstrates that the combination of tafasitamab and rituximab improves efficacy compared to single-agent treatments in models of aggressive B-cell lymphoma in vitro and in vivo.

Unlocking the Power of Complement-Dependent Cytotoxicity: Engineering Strategies for the Development of Potent Therapeutic Antibodies for Cancer Treatments.

The complement system is a crucial part of the innate immune response, providing defense against invading pathogens and cancer cells. Recently, it has become evident that the complement system plays a significant role in anticancer activities, particularly through complement-dependent cytotoxicity (CDC), alongside antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). With the discovery of new roles for serum complement molecules in the human immune system, various approaches are being pursued to develop CDC-enhanced antibody therapeutics. In this review, we focus on successful antibody engineering strategies for enhancing CDC, analyzing the lessons learned and the limitations of each approach. Furthermore, we outline potential pathways for the development of antibody therapeutics specifically aimed at enhancing CDC for superior therapeutic efficacy in the future.

Mapping Antibody Domain Exposure on Nanoparticle Surfaces Using DNA-PAINT.

Decorating nanoparticles with antibodies (Ab) is a key strategy for targeted drug delivery and imaging. For this purpose, the orientation of the antibody on the nanoparticle is crucial to maximize fragment antibody-binding (Fab) exposure and thus antigen binding. Moreover, the exposure of the fragment crystallizable (Fc) domain may lead to the engagement of immune cells through one of the Fc receptors. Therefore, the choice of the chemistry for nanoparticle-antibody conjugation is key for the biological performance, and methods have been developed for orientation-selective functionalization. Despite the importance of this issue, there is a lack of direct methods to quantify the antibodies' orientation on the nanoparticle's surface. Here, we present a generic methodology that enables for multiplexed, simultaneous imaging of both Fab and Fc exposure on the surface of nanoparticles, based on super-resolution microscopy. Fab-specific Protein M and Fc-specific Protein G probes were conjugated to single stranded DNAs and two-color DNA-PAINT imaging was performed. Hereby, we quantitatively addressed the number of sites per particle and highlight the heterogeneity in the Ab orientation and compared the results with a geometrical computational model to validate data interpretation. Moreover, super-resolution microscopy can resolve particle size, allowing the study of how particle dimensions affect antibody coverage. We show that different conjugation strategies modulate the Fab and Fc exposure which can be tuned depending on the application of choice. Finally, we explored the biomedical importance of antibody domain exposure in antibody dependent cell mediated phagocytosis (ADCP). This method can be used universally to characterize antibody-conjugated nanoparticles, improving the understanding of relationships between structure and targeting capacities in targeted nanomedicine.

Abstract 2938: Preclinical development of a novel bispecific mAb-Trap fusion protein, IMM2902, targeting both HER2 and CD47 as cancer immunotherapy

Abstract The overexpression of human epidermal growth factor receptor 2 (HER2) has been identified in a variety of solid tumors including breast cancer (BC), gastric cancer and is associated with tumor recurrence, brain metastasis, and an overall poor prognosis. As such, HER2 has become a major target in the research and development of anticancer drugs. CD47, an integrin-associated protein, is expressed in many human cancer cells, especially upregulated preferentially in HER2-expressing cells. Co-expression of HER2 and CD47 is more frequently detected in recurrent BC patients with poor prognosis when comparing with the counterpart primary tumors. IMM2902 is a first-in-class, clinical-stage, novel recombinant bispecific mAb-Trap fusion protein targeting both CD47 and HER2 simultaneously. The binding activities of IMM2902, measured as EC50, to total CD47+ and HER2+ in BT474, NCI-N87 and SKOV-3 cancer cell lines are 16, 11 and 5 nM respectively. The affinity of IMM2902 to HER2 is about 15 times higher than that of CD47. IMM2902 not only prevents the engagement of CD47 with SIRPα and hence blocks “don’t eat me” signal of CD47/SIRPα, but also induces strong antibody-dependent cell-mediated cytotoxicity (ADCC, EC50 = 0.0045 - 0.0544 nM) and antibody-dependent cellular phagocytosis (ADCP, EC50 = 0.05 - 0.13 nM) activity with specifically engineered IgG1 Fc fragment. Most importantly, IMM2902 induces accelerated HER2 degradation in tumor tissues. Despite its high affinity to CD47, IMM2902 does not binds to CD47 on erythrocyte membrane, therefore, unlike conventional CD47 mAb, IMM2902 demonstrated a minimal impact on red blood cells (RBC). In preclinical research, IMM2902 has demonstrated a much stronger antitumor activity with excellent tolerability in a mouse model of human breast cancer (dose range 3.5 - 10 mg/kg) and a mouse model of human gastric cancer (dose range 1 -18 mg/kg) than that of trastuzumab (targeting HER2 only) alone, IMM01 (a SIRPα-Fc fusion protein) alone, and the combination of trastuzumab and IMM01. In HER2-low SUN-1 xenograft models, IMM2902 has showed a strong tumor growth inhibition at doses between 2 to 18 mg/kg. Intriguingly, IMM2902 also has exhibited potent tumor killing activity against breast cancer resistant to trastuzumab (HCC-1954 and patient-derived xenografts herceptin-resistant model) at doses between 3.5 to 10 mg/kg. Given the strong preclinical antitumor activity as well as the favorable safety profile, IMM2902 may serve as a potent immunotherapy for HER2-expressing cancers via dual blockade of CD47 and HER2. A Phase 1 clinical trial exploring safety, tolerability, and preliminary efficacy of IMM2902 in patients with HER2-expressing advanced solid tumors is currently ongoing in both China (CXSL2101035) and USA (NCT05076591). Citation Format: Binglei Zhang, Song Li, Dianze Chen, Dandan Liu, Huiqin Guo, Chunmei Yang, Li Zhang, Wei Zhang, Xiaoping Tu, Liang Peng, Gui Zhao, Ruliang Zhang, Frank X. Gan, Wenzhi Tian, Fan Zhang, Yongping Song. Preclinical development of a novel bispecific mAb-Trap fusion protein, IMM2902, targeting both HER2 and CD47 as cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2938.

B-Cell Targeting Anti-CD37 Humanized Antibodies Engineered for Potent Effector Functions and Extended Plasma Half-Life

Background: CD37 is a tetraspanin protein abundant on the surface of B-cells but absent in hematopoietic stem cells and plasma cells. Its expression pattern has attracted interest as a therapeutic target for B-cell malignancies including non-Hodgkin lymphomas (NHL), a heterogeneous disease with substantial unmet clinical need due to relapse and resistance to currently available treatments. During the last years, different approaches have been followed to exploit CD37 in targeting malignant B-cells, such as the generation of CAR-T cells and monoclonal antibodies. Of note, the anti-CD37 antibody-drug conjugates (ADC) naratuximab emtansine and the CD37-targeting radioimmunotherapy (RIT) Betalutin have shown efficacy in NHL patients during clinical testing. Here we report on the design of humanized anti-CD37 IgG1 antibodies (NNV029, NNV031 and NNV032) that are equipped with a unique Fc technology and N-glycan engineering for enhanced effector functions and extended plasma half-life. Methods: The humanized anti-CD37 candidates were characterized in vitro and in vivo and benchmarked against the anti-CD20 antibodies approved for NHL treatment, rituximab and obinutuzumab, and a recombinant version of the bi-paratopic anti-CD37 antibody DuoHexaBody-CD37, currently in Phase I clinical development. We assessed their Fcγ receptor binding properties and their ability to induce antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) against CD37-expressing NHL cell lines and patient-derived chronic lymphocytic leukemia (CLL) cells. Moreover, we evaluated their human FcRn binding and transport properties using the human endothelial recycling assay (HERA) followed by studies in human FcRn (Tg32) expressing mice. Lastly, we compared the therapeutic efficacy of the humanized antibodies versus obinutuzumab and recombinant DuoHexaBody-CD37 in a xenograft disseminated Daudi tumor model in Tg32 mice. Results: We revealed that the designed anti-CD37 IgG1 candidates have higher affinity towards FcγRIIa expressed on macrophages and that afucosylation enhanced FcγRIII engagement. Accordingly, using cell-based effector assays, potent ADCP and ADCC were measured. In NHL Burkitt's lymphoma cell lines, ADCP induction by the anti-CD37 humanized antibodies was higher than that displayed by obinutuzumab, rituximab and recombinant DuoHexaBody-CD37, while in DLBCL and MCL cell models it was in a similar range or slightly lower than that shown by the benchmarking antibodies. Moreover, the afucosylated version promoted very strong ADCC, which was superior to that of recombinant DuoHexaBody-CD37 and rituximab, but comparable to that of the afucosylated antibody obinutuzumab. In contrast, all the humanized anti-CD37 IgG1 candidates displayed very modest CDC activation. Fc-engineering for enhanced human FcRn binding was shown to increase human FcRn-mediated rescue from intracellular degradation, which translated into prolonged plasma half-life in Tg32 mice of the anti-CD37 IgG1 antibodies. Finally, all the designed anti-CD37 antibodies performed significantly better than vehicle control treatment in treating Daudi-induced disease in vivo, showing equal therapeutic effect as that of obinutuzumab and recombinant DuoHexaBody-CD37. Conclusions: Taken together, the humanized and Fc-engineered anti-CD37 antibodies show potent effector functions, favorable pharmacokinetic properties and in vivo therapeutic efficacy. As such, the designed CD37 antibodies should be attractive modalities in targeted treatment of B cell malignancies, such as NHL, including lymphomas non-responding to anti-CD20 treatments, but also potentially for B-cell driven autoimmune disorders.

341 (PB121) - MCLA-129, a human anti-EGFR and anti-c-MET bispecific antibody, in patients with advanced NSCLC and other solid tumors: an ongoing phase 1/2 study

Background: MCLA-129, a novel human, EGFR/c-MET bispecific antibody, demonstrates anti-tumor activities in EGFR and/or c-MET driven tumor models via multiple mechanisms, including inhibition of ligand binding, receptor co-degradation, antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated phagocytosis. MCLA-129 is being investigated in patients (pts) with advanced solid tumors in an ongoing First in Human study (NCT04868877). EGFR and c-MET are frequently mutated or overexpressed in epithelial tumors, driving ligand-independent receptor activation.

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.

Abstract 332: Development of a novel therapeutic CLDN18.2 monoclonal antibody and antibody drug conjugate for the treatment of CLDN18.2 positive cancers

Abstract Background: Claudin 18 is a member of the claudin family of tight junction tetraspan cell surface proteins. The 18.2 isoform of claudin 18 (CLDN18.2) is expressed in healthy gastric mucosa cells and largely absent from other normal adult cell types. In contrast to the low expression in healthy tissues, CLDN18.2 is highly overexpressed in many cancer types including gastric and pancreatic malignancies at high frequencies (50-80%). The high tumor-normal expression differential of CLDN18.2 make it an attractive target for both therapeutic monoclonal antibodies (mAbs) and antibody drug conjugate (ADCs). Here, we describe the preclinical development of a novel therapeutic CLDN18.2 mAb and ADC. Materials and Methods: CLDN18.2 specific mAbs were generated through a multiplexed immunization strategy by which mice were immunized with either peptides spanning loop 2 of the CLDN18.2 extracellular domain or mouse NIH3T3 cells overexpressing the full length CLDN18.2 protein. Antibody clones were screened by flow cytometry for selective binding to cell surface CLDN18.2. A CLDN18.2-directed ADC was generated from the fully humanized clinical candidate CLDN18.2 mAb by MMAE conjugation with a cleavable linker. CLDN18.2 positivity in cell line (CDX) and patient derived xenograft (PDX) models was determined by IHC assay. Results: Selective binding of the CLDN18.2-mAb to CLDN18.2, without cross reactivity to the more widely expressed CLDN18.1 isoform, was confirmed in human cancer cell lines and cells engineered to overexpress each isoform. The CLDN18.2-mAb demonstrated strong induction of both antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) in in vitro assays; suggesting that Fc effector function is a key component of the mechanism of action of the mAb. Treatment of CLDN18.2 positive gastric and pancreatic CDXs with CLDN18.2-mAb monotherapy induced significant inhibition of tumor growth in CD-1 nude mice. Binding of CLDN18.2-mAb or CLDN18.2-ADC to the cell surface CLDN18.2 induced internalization of the protein-antibody complex. Complete regression of CDX tumors was observed in response to treatment with the CLDN18.2-ADC in three separate CLDN18.2 positive models (two pancreas, one gastric). Moreover, complete loss of xenograft tumor burden was also observed in 4/5 CLDN18.2 positive pancreas PDX models. No impact on xenograft tumor growth as observed in response to treatment with either the CLDN18.2-mAb or CLDN18.2-ADC in CLDN18.2 negative CDX/PDX models. Discussion: These data support the clinical development of this CLDN18.2-mAb and CLDN18.2-ADC for the treatment of CLDN18.2 positive cancers. Each of these molecules will begin phase 1 clinical testing in early 2022. Citation Format: Neil A. O'brien, Martina S. McDermott, Jun Zhang, Ke Wei Gong, Ming Lu, Benjamin G. Hoffstrom, Dylan Conklin, Tong Luo, Kevin Chau, Min Liang, Timothy R. Donahue, John A. Glaspy, Leonard Presta, Dennis J. Slamon. Development of a novel therapeutic CLDN18.2 monoclonal antibody and antibody drug conjugate for the treatment of CLDN18.2 positive cancers [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 332.

Abstract P2-08-09: Investigation of autoantibodies (AAbs) in HER2+ breast cancer (BC) patients treated in the neo-adjuvant setting

Abstract Background: HER2+ breast cancer is treated with anti-HER2 IgG monoclonal antibody therapies such as trastuzumab and pertuzumab. These antibodies inhibit HER2-driven intracellular signaling and can also be described as a form of passive immunotherapy, engaging effector immune cells in antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). Tumor-associated autoantibodies (AAbs) to tumor neo-antigens can be generated by the adaptive immune response before, or in response to, therapy. There is growing evidence of a functional role for tumor-associated AAbs in the immune response to tumors, with IgG and IgA AAbs capable of mediating ADCC and ADCP. Using the innovative Nucleic Acid-Programmable Protein Array (NAPPA) platform we examined the levels and targets of circulating AAbs to ~1700 human proteins before, during and after treatment in HER2+ breast cancer treated in the neo-adjuvant setting. . Methods: Pre-treatment, On-treatment (pre- Cycle 2) and Post-treatment (post-Cycle 6) serum samples were obtained from HER2+ breast cancer patients (n=19) treated on the ICORG 10-05 clinical trial. Patients were treated with neo-adjuvant chemotherapy (docetaxel (T)/carboplatin (C)) +/- trastuzumab (H), lapatinib (L) or HL, TCH n=9, TCHL n= 7, TCL n=3. Patients were classified as having a pathological complete response (pCR, n=6), a partial response (PR, n=6) or a non-response (NR, n=7) to treatment. IgG and IgA autoantibody levels were determined using the HD-NAPPA system, focussing on a subset of ~ 1700 human protein targets. A normalised intensity score of &gt 5 was utilised as a cut-off for detection. Results: 92 IgG AAbs and 29 IgA AAbs were detected in all samples, across all timepoints (n=57). When IgG AAbs were divided by patient response, 26 AAbs unique to pCR, 26 AAbs unique to PR and 16 AAbs unique to NR were detected. 5 IgG AAbs (BCL11B, MAP6, PRDM8, SYTL2, TP53BP2) were common to all HER2+ breast cancer patients. When IgA AAbs were divided by patient response, 11 AAbs unique to pCR, 6 AAbs unique to PR and 7 AAbs unique to NR were detected. 2 IgA AAbs (TDP1 and MAP6) were common to all HER2+ breast cancer patients. Treatment-induced AAbs were detected in On-treatment and Post- treatment samples in 16/19 patients examined. Conclusions: Our results suggest HER2+ BC patients have unique circulating AAbs associated with treatment response and AAb levels change in response to treatment. Further investigation of AAb levels as biomarkers of response in HER2+ BC is warranted in a larger cohort of patients. Citation Format: Denis Martin Collins, Ji Qiu, Jaine Blayney, Nuala McCabe, Richard Kennedy, Joshua LaBaer, John Crown. Investigation of autoantibodies (AAbs) in HER2+ breast cancer (BC) patients treated in the neo-adjuvant setting [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-08-09.

Antibody-Dependent THP-1 Cell-Mediated Phagocytosis of Plasmodium falciparum-Infected Erythrocytes.

Antibodies that recognize variant surface antigens (VSAs) expressed on Plasmodium falciparum-infected erythrocytes (IEs) opsonize IEs for phagocytic clearance. The anti-VSA antibodies promote antibody-dependent cellular phagocytosis (ADCP) of IEs by interacting with innate immune cells. ADCP is an important immune effector mechanism of parasite clearance. ADCP can be a tool to assess the efficacy of vaccine-induced antibodies, in addition to measuring the neutralizing ability of antibodies. Here, we developed and validated an efficient and high-throughput plate-based flow cytometric assay to measure ADCP of IEs using the human monocytic THP-1 cell line. This flow cytometric assay can be used to analyze the level of naturally acquired or vaccine-induced opsonic antibodies in large cohorts.

Development of a novel reporter gene assay to evaluate antibody-dependent cellular phagocytosis for anti-CD20 therapeutic antibodies

More than 100 monoclonal antibodies (mAbs) have been approved by FDA. The mechanism of action (MoA) involves in neutralization of a specific target via the Fab region and Fc effector functions through Fc region, while the latter include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). ADCP has been recognized one of the most important MoAs, especially for anti-cancer mAbs in recent years. However, traditional bioassays measuring ADCP always introduced primary macrophages and flow cytometry, which are difficult to handle and highly variable. In this study, we engineered a monoclonal Jurkat/NFAT/CD32a-FcεRIγ effector cell line that stably expresses CD32a-FcεRIγ chimeric receptor and NFAT-controlled luciferase. The corresponding mAb could bind with the membrane antigens on the target cells with its Fab fragment and CD32a-FcεRIγ on the effector cells with its Fc fragment, leading to the crosslinking of CD32a-FcεRIγ and the resultant expression of subsequent NFAT-controlled luciferase, which represents the bioactivity of ADCP based on the MoA of the mAb. With rituximab as the model mAb, Raji cells as the target cells, and Jurkat/NFAT/CD32a-FcεRIγ cells as the effector cells, we adopted the strategy of Design of Experiment (DoE) to optimize the bioassay. Then we fully validated the established bioassay according to ICH-Q2(R1), which proved the good assay performance characteristics of the bioassay, including specificity, accuracy, precision, linearity, stability and robustness. This RGA can be applied to evaluate the -ADCP bioactivity for anti-CD20 mAbs in lot release, stability testing as well as biosimilar comparability. The engineered cells may also potentially be used to evaluate the ADCP bioactivity of mAbs with other targets.

ABCL-021: FRONT-MIND: A Phase III, Randomized, Double-Blind, Placebo-Controlled Study Comparing Efficacy and Safety of Tafasitamab + Lenalidomide + R-CHOP vs R-CHOP Alone for Newly-Diagnosed High-Intermediate and High-Risk Diffuse Large B-Cell Lymphoma (DLBCL)

<h3>Context</h3> Tafasitamab is an Fc-modified, humanized anti-CD19 monoclonal antibody, which induces enhanced <i>in vitro</i> antibody-dependent cell-mediated cytotoxicity and phagocytosis. Preclinical studies data suggest that tafasitamab+lenalidomide may synergistically enhance cytotoxicity to malignant B-cells. Tafasitamab+lenalidomide is FDA-approved in adult patients with relapsed/refractory DLBCL who are ineligible for autologous stem cell transplantation. R-CHOP is the current standard of care (SOC) for patients with newly-diagnosed DLBCL. Data from the Phase Ib FIRST-MIND (NCT04134936) study in newly-diagnosed DLBCL patients suggest that tafasitamab+lenalidomide when added to R-CHOP is tolerable in patients with treatment-naïve DLBCL. <h3>Objective</h3> To assess the efficacy and safety of tafasitamab+lenalidomide+R-CHOP <i>vs</i> R-CHOP alone in previously untreated, high-intermediate and high-risk patients with DLBCL. <h3>Design</h3> FRONT-MIND is a Phase III, multicenter, randomized, double-blind, placebo-controlled study. Patients will be followed-up for up to 60 months after end of treatment. <h3>Setting</h3> Patients will be enrolled from approximately 350 centers in America, Europe, and Asia-Pacific. <h3>Patients</h3> Eligible patients (n=880) will be aged 18–80 years with previously untreated local biopsy-proven, CD20-positive DLBCL (IPI ≥3 if >60 years/age-adjusted IPI 2–3 if ≤60 years), and ECOG PS 0–2. Patients with transformed lymphoma (except double or triple hit lymphoma) are excluded. <h3>Interventions</h3> Patients will be randomized to receive six 21-days (D) cycles of tafasitamab (12 mg/kg intravenous [IV], D 1, 8, and 15)+lenalidomide (25 mg orally, D1–10)+R-CHOP or six 21-D cycles of tafasitamab placebo (D1, 8, and 15)+lenalidomide placebo (orally, D1–10)+R-CHOP. <h3>Main Outcome Measures</h3> The primary endpoint is investigator-assessed progression-free survival. Secondary endpoints include investigator-assessed event-free survival, overall survival, and safety. <h3>Results</h3> Not yet available. <h3>Conclusions</h3> As 30–50% of patients overall relapse or are refractory to first-line therapy, there remains a high unmet need to improve treatment options for newly diagnosed patients, particularly those with high-risk DLBCL. The combination of tafasitamab, lenalidomide, and R-CHOP may have synergistic potential. Preliminary data from the FIRST-MIND study suggest that tafasitamab±lenalidomide+R-CHOP is tolerable in patients with treatment-naïve DLBCL. The present study will provide further evaluation of clinical benefits and safety when adding tafasitamab+lenalidomide to current SOC in newly diagnosed patients with high-intermediate and high-risk DLBCL treated with R-CHOP.

Protease-Activation of Fc-Masked Therapeutic Antibodies to Alleviate Off-Tumor Cytotoxicity.

The interaction of the Fc region of therapeutic antibodies and antibody-drug conjugates with Fcγ receptors (FcγRs) can lead to unpredictable and severe side effects. Over the last decades several strategies have been developed to overcome this drawback, including extensive Fc- and glycoengineering and antibody isotype switching. However, these approaches result in permanently Fc-silenced antibody derivates which partially or completely lack antibody-mediated effector functions. Nevertheless, for a majority of antibody-based drugs, Fc-mediated effector functions, like antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) as well as complement-dependent cytotoxicity (CDC), represent the most substantial modes of action. We argued that a new strategy combining the beneficial properties of Fc-silencing and controlled activation of effector functions can pave the way to potent antibody therapeutics, reducing the FcγRs-mediated off-target toxicity. We present a novel Fc-tamed antibody format, where the FcγR-binding sites of antibodies are blocked by anti-isotypic masking units, hindering the association of FcγR and complement component 1 (c1q) to the Fc domain. The masking units were genetically fused to trastuzumab, including a protease-addressable peptide-liker. Our Fc-tamed antibodies demonstrated completely abolished interaction to soluble high-affinity Fcγ-Receptor I and c1q. In reporter cell-based ADCC assays, our Fc-tamed antibodies exhibited a 2,700 to 7,100-fold reduction in activation, compared to trastuzumab. Upon demasking by a tumor-associated protease, the Fc-activated antibodies demonstrated restored FcγR-binding, c1q-binding and the ability to induce potent ADCC activation. Furthermore, cell killing assays using donor-derived NK cells were performed to validate the functionality of the Fc-tamed antibody variants. To our knowledge, this approach represents the first non-permanently Fc-silenced antibody, which can be re-activated by a tumor-associated protease, eventually extending the field of novel antibody formats.

Abstract 952: The bispecific antibody MCLA-129 impairs NSCLC tumor growth by targeting EGFR and c-MET, inhibiting ligand-induced signaling and promoting ADCC and ADCP

Abstract MCLA-129 is an ADCC-enhanced common light chain bispecific human IgG1 Biclonics® antibody specifically targeting the receptor tyrosine kinases EGFR and c-MET. It inhibits activation and downstream signaling of EGFR and c-MET induced by their respective ligands EGF and HGF and promotes elimination of tumor cells via Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Antibody-Dependent Cell-Mediated Phagocytosis (ADCP). MCLA-129 was developed to overcome c-MET signaling-dependent EGFR TKI-resistance mechanisms and was identified during an unbiased screening campaign with a focus on inhibition of ligand-induced proliferation and migration of cancer cells. Selectivity of MCLA-129 for tumor cells is achieved by simultaneously targeting both EGFR and c-MET. MCLA-129 blocks EGF and HGF binding to EGFR and c-MET, respectively, and consequently inhibits their ligand-induced phosphorylation. MCLA-129 was shown to bind critical residues on EGFR and c-MET for EGF and HGF ligand binding. GlymaxX® low fucose glycoengineering technology was used to enhance the mononuclear cell ADCC activity of the bispecific antibody. MCLA-129 demonstrated potent dose-dependent ADCC and ADCP against all NSCLC cell lines tested, which positively correlated with the EGFR and c-MET expression levels on the target cells. Significant inhibition of tumor growth of NCI-H1975EGFR L858R, T790M and HCC827/ER1EGFR del (E746, A750), c-MET amplified NSCLC cell line derived tumors was observed following MCLA-129 treatment. MCLA-129 can overcome HGF-mediated EGFR-TKI resistance, as demonstrated by testing a non-ADCC enhanced version of this antibody in a panel of NSCLC cell lines in vitro and orthotopic tumors in vivo showing that it inhibits EGFR and c-MET activity. Significant tumor regression was observed following treatment of immunodeficient xenograft NSG-hHGFki mice bearing orthotopic tumors established from HCC827EGFR del (E746, A750) cells. In addition, in an acquired erlotinib EGFR TKI resistance model, treatment of mice harboring tumors as big as &amp;gt;500 mm3 led to significant reduction of tumor size which persisted after the treatment period. Taken together these data demonstrate that MCLA-129 is a potent inhibitor of tumor growth applying various mechanisms of action, including inhibition of c-MET and EGFR signaling, ADCC and ADCP. MCLA-129 holds promise as a potential treatment for patients with NSCLC and other cancers, and warrants clinical evaluation. Citation Format: David J. de Gorter, Alexandre Deshiere, Martijn van Rosmalen, Christian Wohn, Berina Eppink, Tristan Gallenne, Rinse Klooster, Li Mao, Wenxin Xu, Liang Deng, Qingyu Shu, Wei Liu, John de Kruif, Mario Di Matteo, Massimiliano Mazzone, Mark Throsby, Cecile A. Geuijen. The bispecific antibody MCLA-129 impairs NSCLC tumor growth by targeting EGFR and c-MET, inhibiting ligand-induced signaling and promoting ADCC and ADCP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 952.

Single-Arm Phase II Study of MOR208 Combined with Lenalidomide in Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma: L-Mind

Single-Arm Phase II Study of MOR208 Combined with Lenalidomide in Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma: L-Mind