Antibody effector functions including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) are mediated through the interaction of the antibody Fc region with Fcγ receptors present on immune cells. Several approaches have been used to modulate antibody Fc-Fcγ interactions with the goal of driving an effective antitumor immune response, including Fc point mutations and glycan modifications. However, robust antibody-Fcγ engagement and immune cell binding of Fc-enhanced antibodies in the periphery can lead to the unwanted induction of systemic cytokine release and other dose-limiting infusion-related reactions. Creating a balance between effective engagement of Fcγ receptors that can induce antitumor activity without incurring systemic immune activation is an ongoing challenge in the field of antibody and immuno-oncology therapeutics. Herein, we describe a method for the reversible chemical modulation of antibody-Fcγ interactions using simple poly(ethylene glycol) (PEG) linkers conjugated to antibody interchain disulfides with maleimide attachments. This method enables dosing of a therapeutic with muted Fcγ engagement that is restored in vivo in a time-dependent manner. The technology was applied to an effector function enhanced agonist CD40 antibody, SEA-CD40, and experiments demonstrate significant reductions in Fc-induced immune activation in vitro and in mice and nonhuman primates despite showing retained efficacy and improved pharmacokinetics compared to the parent antibody. We foresee that this simple, modular system can be rapidly applied to antibodies that suffer from systemic immune activation due to peripheral FcγR binding immediately upon infusion.

Supplementary Fig. S1 from Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index

Abstract There are four vedotin drug linker containing antibody drug conjugates currently approved for treating cancer patients, proving that this technology platform is an effective modality for the treatment of both hematologic and solid tumors. The primary mechanism of vedotin ADCs, direct cytotoxicity, is the result of intracellular release of monomethyl auristatin E (MMAE) after cleavage of the valine-citrulline (Val-Cit) dipeptide by lysosomal proteases. However, off-tumor delivery of MMAE can result in dose limiting bone marrow toxicity in preclinical models. Maintaining potency in cancer cells while reducing the bone marrow toxicity of vedotin ADCs could allow higher or more frequent clinical dosing and potentially improved response rates and patient outcomes. To decrease bone marrow toxicity of MMAE empowered ADCs, we developed a process to identify peptidic linker systems that provide selectivity for cancer cells over bone marrow cells. From this process we identified a tripeptide sequence (D-leucine-alanine-glutamate, dLAE) that is preferentially cleaved in cancer cells and used it to develop a drug linker that releases MMAE upon cleavage of this tripeptide. Here, we present in vitro and in vivo data comparing ADCs containing vedotin and dLAE tripeptide drug linkers. While ADCs with the dLAE drug linker had comparable in vitro cytotoxicity and antitumor activity to vedotin, the dLAE ADCs had a higher maximum tolerated dose in exploratory rat and cynomolgus macaque toxicity studies due to a reduction in bone marrow toxicity. Reduced bone marrow toxicity was correlated with decreased linker catabolism in bone marrow cells for conjugates bearing the dLAE tripeptide compared to Val-Cit, while similar catabolism was observed in cancer cells. Together, these preclinical data highlight that changing the peptide of the vedotin drug linker from Val-Cit to dLAE can yield ADCs with an improved preclinical therapeutic window. Given the preclinical characteristics of the dLAE drug linker, a clinical trial is being planned to evaluate an anti-CD30 ADC (SGN-35T). Citation Format: Noah A Bindman, Roma Yumul, Erica E McKinney, Scott Blackburn, Nicole Blesie, Xinqun Zhang, Brendan Drouhard, Sarah Anderson, Calvin Neace, David Ortiz, KC Crowder, Nancy Everds, Chris Carosino, Forgivemore Magunda, Django Sussman, Esther Trueblood, Nicole M. Okeley, Peter Senter. Discovery of a novel auristatin antibody drug conjugate drug linker with equal efficacy and reduced bone marrow toxicity compared to vedotin [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C113.

Supplementary Table S4 from Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index

Supplementary Fig. S2 from Anti-CD30 diabody-drug conjugates with potent antitumor activity

Supplementary Table S3 from Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index

Modification of effector function has proven to be an effective modality for optimizing activity and tolerability of therapeutic antibodies. Currently available methods to modulate effector function include the introduction of point mutations in the Fc region and glycan engineering of the antibody. Here we present an alternative and complementary method of tuning effector function utilizing a conjugation-based approach. This methodology uses conjugation of polyethylene glycol (PEG) to native cysteines of an antibody to impair FcγR binding of antibodies to innate immune effector cells. Utilizing maleimide or disulfide conjugation techniques, attenuated effector function can be either permanent or restored over time through a de-conjugation process. Impacts of PEGylation on FcγR binding, signaling, and restoration of function were assessed in vitro and in vivo. As a proof-of-concept, the lead technology was applied to an agonist CD40 antibody, which resulted in significant reductions in systemic cytokine production in hCD40 mice and non-human primates, while demonstrating retained efficacy and improved pharmacokinetics. Additionally, we combined the conjugation technology with glycan engineering and FcγR enhancing point mutations to impart unique effector function profiles to clinical antibodies. This simple, modular approach can be rapidly applied to existing antibodies to reduce immune-driven toxicities, such as infusion reactions, and optimize effector function activity. Citation Format: Philip N. Moquist, Chris I. Leiske, Noah A. Bindman, Xinqun Zhang, Nicole Duncan, Weiping Zeng, Serena W. Wo, Abbie Wong, Clark M. Henderson, Karalyne Crowder, Haley D. Neff-LaFord, Django Sussman, Shyra J. Gardai, Matthew R. Levengood. Reversible chemical modification of antibodies: A complementary approach to tuning FcγR binding that maintains anti-tumor activity while mitigating peripheral immune activation [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 2656.

Supplementary Table 1: Surface antigen density impacts in vitro potency of anti-LIV-1 ADC . MCF-7 ATCC cells from three sources with varying levels of LIV-1 expression were tested with SGN-LIV1A for cytotoxicity. Lower antigen density resulted in decreased potency.

Supplementary Fig. S2 from Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index

Supplementary Material from Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index

Gating strategy, primary AML cytotoxicity assay,additional cell liens for pH2AX assay and caspase assay, and h7G3ec antibody sequence

Supplementary Fig. S1 from Anti-CD30 diabody-drug conjugates with potent antitumor activity

Supplementary Material from Anti-CD30 diabody-drug conjugates with potent antitumor activity

Background Antibody effector functions including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) are mediated through the interaction of the antibody Fc region with Fc gamma receptors present on immune cells. Several approaches have been used to modulate antibody Fc-Fc gamma receptor interactions with the goal of driving an effective antitumor immune response. One such approach is removal of fucose on the antibody core glycan to increase binding to Fc gamma receptor IIIa (CD16a) and drive increased ADCC and immune agonism. However, robust antibody Fc engagement and immune cell binding of non-fucosy-lated antibodies in the periphery can lead to unwanted induction of systemic cytokine release and other dose-limiting infusion-related reactions. Identifying a balance between effective engagement of Fc gamma receptors that can induce antitumor activity without incurring systemic immune activation is an ongoing challenge in the field of antibody and immuno-oncology therapeutics. Methods A method for the reversible modulation of antibody Fc interactions was designed and applied to a series of non-fucosylated antibodies. This methodology utilizes chemical conjugation of polyethylene glycol (PEG) linkers to the interchain disulfides of an antibody to initially impair binding to Fc gamma receptors on peripheral cells upon administration but allow for restoration of antibody effector function through de-conjugation over time. Impacts of PEGylation on Fc gamma receptor binding, signaling, and restoration of function were assessed in vitro and in vivo. Results In vitro binding and signaling assays with various loaded conjugates were used to identify a preferred linker for-mat that reduces binding and activity of non-fucosylated antibodies

Ladiratuzumab vedotin (LV, SGN-LIV1A) is an antibody-drug conjugate directed against the LIV-1 protein that is currently under investigation for treatment in metastatic breast cancer. This ADC consists of a monoclonal antibody conjugated to monomethyl auristatin E (MMAE), a potent microtubule-disrupting agent. LV antitumor activity is thought to primarily be the result of intracellular payload release, leading to mitotic arrest and apoptotic cell death. While LV-induced cell death has been extensively studied, its potential immune modulatory activity has yet to be explored. Whereas normal apoptosis is nonimmunogenic, multiple chemotherapeutics have been shown to elicit a unique form of immunogenic cell death (ICD), characterized by the exposure of various proinflammatory intermediates prior to cell death. We have recently demonstrated that a CD30-directed ADC containing MMAE (brentuximab vedotin) was able to elicit ICD as a consequence of microtubule disruption and subsequent induction of endoplasmic reticulum stress. Induction of ICD in preclinical models of Hodgkin lymphoma results in directed antitumor immune responses, which is further potentiated by anti-PD-1 therapy. In this study, we provide evidence that ladiratuzumab vedotin induces apoptosis of tumor cells in a manner that is consistent with ICD. Treatment of LIV-1+ cells with LV resulted in the activation of all arms of the ER stress response, via the activation of ATF6 and phosphorylation of IRE1 and eIF2a. Severity of ER stress was confirmed by phosphorylation of the downstream effector Jun N-terminal kinase (JNK), and induction of the unfolded protein response. Critically, induction of C/EBP homologous protein (CHOP) indicated irreparable ER stress and subsequent apoptosis. Concurrent with ER stress induction but prior to cell death, LV-treated cells also released ATP and HMGB1 into the supernatant, hallmarks of ICD that are important for immune cell activation and recruitment into the tumor microenvironment. Critically, treatment of subcutaneously engrafted LIV1+ MCF7 cells generated a proinflammatory cytokine response within the tumor. Furthermore, intratumoral dendritic cells and macrophages displayed an inflammatory phenotype, highlighted by increased MHCII expression, providing evidence for improved immune activation and engagement. Via induction of ER stress, tumor cells killed by ladiratuzumab vedotin may initiate an antitumor immune response and provide a rationale for exploring therapeutic strategies that combine ADCs with other immune stimulatory regimens. Citation Format: Anthony T. Cao, Shaylin Higgins, Nicole Stevens, Shyra J. Gardai, Django Sussman. Additional mechanisms of action of ladiratuzumab vedotin contribute to increased immune cell activation within the tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2742.

Abstract B cell maturation antigen (BCMA) has recently emerged as an attractive therapeutic target in multiple myeloma. BCMA has restricted expression on plasma cells with little to no expression on other normal tissues, but is upregulated on the surface of multiple myeloma cells. BCMA can regulate proliferation and survival of myeloma cells via binding to its ligands APRIL and BAFF and induce downstream signaling pathways. Thus, several approaches to target BCMA are currently under clinical investigation, including chimeric antigen receptor (CAR) T cell therapies, bispecific antibodies and antibody drug conjugates. The Antibody-Coupled T cell Receptor (ACTR) technology is a universal, engineered T cell therapy consisting of the extracellular domain of human CD16 and the intracellular T cell co-stimulatory and signaling domains. ACTR is designed to engage the Fc domain of therapeutic antibodies opsonized to target cells to mediate anti-tumor activity. Previous work has demonstrated ACTR T cell activity in combination with rituximab, trastuzumab, and hu14.18 K322A against CD20, Her2, and GD2 expressing cell lines, respectively (Kudo et al. Cancer Res 2014; 74:93-103). Currently ACTR is being evaluated in Phase I clinical trials with rituximab to treat relapsed refractory B cell lymphoma. Here we demonstrate a humanized afucosylated anti-BCMA antibody, SEA-BCMA, binds to ACTR expressing T cells with high affinity and mediates T cell activation, potent cytotoxicity, cytokine release and proliferation across a wide range of BCMA expressing myeloma cells. ACTR activity was specific to SEA-BCMA - opsonized target cells, dose dependent and had no activity on BCMA negative tumor lines. Furthermore, the SEA-BCMA antibody has additional properties that might contribute to a therapeutic effect, including blocking the binding of ligands to BCMA and driving natural killer cell mediated ADCC effects. These preclinical studies demonstrate a promising multi-faceted activity of ACTR T cells in combination with the anti-BCMA antibody, SEA-BCMA, for clinical consideration in multiple myeloma patients. Citation Format: Tooba Cheema, Taylor Hickman, Katie O'Callaghan, Lori Westendorf, Luke Manlove, Shyra Gardai, Allison Nelson, Ryan Boomer, Kathleen McGinness, Birgit Schultes, Seth Ettenberg, Django Sussman, Heather Huet. Efficient targeting of BCMA-positive multiple myeloma cells by antibody-coupled T-cell receptor (ACTR) engineered autologous T cells in combination with an anti-BCMA antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4605. doi:10.1158/1538-7445.AM2017-4605

Abstract Glypican-3 (GPC3) is a GPI-anchored member of the heparan sulfate proteoglycan family. GPC3 is an oncofetal antigen expressed transiently during fetal development with re-expression during malignant transformation. GPC3 is an ideal tumor target as expression has been found in numerous epithelial malignancies, with highest expression in hepatocellular carcinoma (HCC) and non-small cell lung carcinoma (NSCLC), and normal tissue expression is highly restricted (Baumhoer D., Am J Clin Pathol, 2008.). Adoptive T-cell therapy with single-chain variable fragment (scFv)-derived chimeric antigen receptors (CARs) has transformed cancer therapy, but the broad applicability of this approach has been limited in part by safety concerns due the constitutive expression of a biologically active targeting receptor. The Antibody-Coupled T-cell Receptor (ACTR) platform is a universal, engineered T-cell therapy designed to engage the Fc domain of therapeutic antibodies opsonized to tumor cells to mediate anti-tumor activity. ACTR activity is therefore both regulatable and flexible, providing enhanced therapeutic control and improved safety of the T cell therapy. Using both HCC and NSCLC tumor cell lines, we tested a panel of wild-type and afucosylated antibodies with similar binding affinities that bound to regions spanning the GPC3 protein across unique epitopes. We found that for GPC3 targeting antibodies, the greatest activity in a Jurkat-NFAT reporter assay was observed for the afucosylated antibody that bound GPC3 most proximal to the membrane. Further, the antibody that bound membrane proximal also had the most potent activity in primary ACTR T cell cytotoxicity and cytokine release assays. The physical distance between T cells and tumor targets has been previously determined to impact T cell activation for both peptide-MHC and CAR-T interactions. Similarly, our results demonstrate a potential relationship between spatial distance of tumor targets and T cells in determining the activity ACTR transduced T cells when targeting GPC3. Our data demonstrate that ACTR T cell activity is antibody-specific and dose-titratable, highlighting both efficacy and improved safety of the ACTR T cell platform when targeting GPC3+ solid tumor malignancies. Citation Format: Greg Motz, John Shin, Kathleen Whiteman, Birgit Schultes, Tapasya Pai, Lori Westendorf, Seth Ettenberg, Travis Biechele, Django Sussman, Heather Huet. Superior T cell activity of a membrane-proximal binding antibody when targeting Glypican-3 with an antibody-coupled T-cell receptor (ACTR) armed T cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3762. doi:10.1158/1538-7445.AM2017-3762

Abstract Treatment choices for acute myelogenous leukemia (AML) patients resistant to conventional chemotherapies are limited and novel therapeutic agents are needed. IL3 receptor alpha (IL3Rα, or CD123) is expressed on the majority of AML blasts, and there is evidence that its expression is increased on leukemic relative to normal hematopoietic stem cells, which makes it an attractive target for antibody-based therapy. Here, we report the generation and preclinical characterization of SGN-CD123A, an antibody–drug conjugate using the pyrrolobenzodiazepine dimer (PBD) linker and a humanized CD123 antibody with engineered cysteines for site-specific conjugation. Mechanistically, SGN-CD123A induces activation of DNA damage response pathways, cell-cycle changes, and apoptosis in AML cells. In vitro, SGN-CD123A–mediated potent cytotoxicity of 11/12 CD123+ AML cell lines and 20/23 primary samples from AML patients, including those with unfavorable cytogenetic profiles or FLT3 mutations. In vivo, SGN-CD123A treatment led to AML eradication in a disseminated disease model, remission in a subcutaneous xenograft model, and significant growth delay in a multidrug resistance xenograft model. Moreover, SGN-CD123A also resulted in durable complete remission of a patient-derived xenograft AML model. When combined with a FLT3 inhibitor quizartinib, SGN-CD123A enhanced the activity of quizartinib against two FLT3-mutated xenograft models. Overall, these data demonstrate that SGN-CD123A is a potent antileukemic agent, supporting an ongoing trial to evaluate its safety and efficacy in AML patients (NCT02848248). Mol Cancer Ther; 17(2); 554–64. ©2017 AACR.

Abstract Antibody Drug Conjugates (ADCs) combine the specificity of antibody therapies with the potency of highly toxic payloads and have become an established therapeutic tool for the treatment of multiple types of cancer. The design process for novel ADCs requires careful attention to the biological target and chemotype selection. Increasingly, the importance of linker attachment and location is also recognized as a critical variable for optimal therapeutic efficacy. For ADCs employing cysteine conjugation, the payload to be delivered has commonly been attached to the antibody backbone via reaction with the native cysteines of the inter-chain disulfides. These cysteines are found in the constant domain of the light chains, the CH1 domain and hinge region of the heavy chains. Alternatively, the chemotype can be conjugated to discrete locations on the antibody backbone by engineering cysteine mutations into the protein sequence, allowing for conjugation in a site-specific manner. For certain chemotypes, site-specific conjugation has demonstrated several distinct advantages over native disulfide conjugation, including homogenous ADC loading, malemide attachment stability and hydrophobic masking of the payload. These effects are particularly applicable to conjugates made at mutation site S239C, which is found on the interior face of the CH2 of the Fc region. Moreover, we have recently discovered that ADCs using the antimitotic tubulysin M show greatly improved chemotype stability when conjugated specifically to the S239C site. In plasma, the tubulysin M molecule is susceptible to the loss of an acetate side chain on the tubuvaline subunit, a change which greatly diminishes tubulin binding and cytotoxicity. Using ex vivo methods and mass spectroscopy we show that, compared to tubulysin M conjugates made to the native interchain disulfide bonds, conjugation to S239C protects the lability of this acetate group and demonstrates improved in vitro cytotoxicity following ADC plasma incubation. With the aim of identifying further protective conjugation locations, we generated and analyzed 20 additional cysteine mutation sites predicted to share similar biophysical properties to S239C. Top performing single mutations were further combined with S239C and evaluated for a combined effect on tubulysin M acetate stability. Surprisingly, several of the new cysteine mutation sites demonstrate a greater effect on tubulysin M stability when used in conjunction with S239C than when used alone. The results on acetate stability following plasma incubation are further reflected by enhanced cytotoxic potency in vitro using pre incubated tubulysin M ADCs. This data demonstrates that appropriate site-specific conjugation can protect against chemotype degradation, and in addition to the advantages of hydrophobic masking and malemide stability, these findings underline the importance of location of chemotype conjugation for optimal ADC design and engineering. Citation Format: Andrew Waight, Chris Leiske, Travis Biechele, Patrick Burke, Rory Rohm, Peter Senter, Dennis Benjamin, Django Sussman. Cysteine mutant location affects chemotype lability in site-specific antibody drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-066. doi:10.1158/1538-7445.AM2017-LB-066