Reversible Chemical Modification of Antibody Effector Function Mitigates Unwanted Systemic Immune Activation.

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.

Nonfucosylation of an anti-TIGIT antibody enhances FcγR engagement, driving innate immune activation and antitumor activity.

TIGIT is an immune checkpoint receptor expressed on activated and memory T cells, immunosuppressive T regulatory cells, and natural killer (NK) cells. TIGIT has emerged as an attractive target for antitumor therapies, due to its proposed immunosuppressive effects on lymphocyte function and T cell activation. We generated an anti-TIGIT monoclonal antibody (mAb) that binds with high affinity to human, non-human primate, and murine TIGIT and through multiple experimental methodologies demonstrated that checkpoint blockade alone is insufficient for antitumor activity. Generating anti-TIGIT mAbs with various Fc backbones we show that muting the Fc-Fcγ receptor (FcγR) interaction failed to drive antitumor activity, while mAbs with Fc functional backbones demonstrate substantial antitumor activity, mediated through activation of antigen-presenting cells (APCs), T cell priming, and NK-mediated depletion of suppressive Tregs and exhausted T cells. Further, nonfucosylation of the Fc backbone resulted in enhanced immune responses and antitumor activity relative to the intact IgG1 backbone. The improved activity correlated with the biased FcγR interaction profile of the nonfucosylated anti-TIGIT mAb, which supports that FcγRIIIa binding with decreased FcγRIIb binding favorably activates APCs and enhances tumor-specific CD8+ T cell responses. The anti-TIGIT mAbs with intact FcγR interacting backbones also demonstrated synergistic enhancement of other standard antitumor treatments, including anti-PD-1 treatment and a model monomethyl auristatin E antibody-drug conjugate. These findings highlight the importance of the anti-TIGIT mAb's Fc backbone to its antitumor activity and the extent to which this activity can be enhanced through nonfucosylation of the backbone.

SGN-B7H4V, an investigational vedotin ADC directed to the immune checkpoint ligand B7-H4, shows promising activity in preclinical models.

BACKGROUND: SGN-B7H4V is a novel investigational vedotin antibody-drug conjugate (ADC) comprising a B7-H4-directed human monoclonal antibody conjugated to the cytotoxic payload monomethyl auristatin E (MMAE) via a protease-cleavable maleimidocaproyl valine citrulline (mc-vc) linker. This vedotin linker-payload system has been clinically validated in multiple Food and Drug Administration approved agents including brentuximab vedotin, enfortumab vedotin, and tisotumab vedotin. B7-H4 is an immune checkpoint ligand with elevated expression on a variety of solid tumors, including breast, ovarian, and endometrial tumors, and limited normal tissue expression. SGN-B7H4V is designed to induce direct cytotoxicity against target cells by binding to B7-H4 on the surface of target cells and releasing the cytotoxic payload MMAE upon internalization of the B7-H4/ADC complex. METHODS: B7-H4 expression was characterized by immunohistochemistry across multiple solid tumor types. The ability of SGN-B7H4V to kill B7-H4-expressing tumor cells in vitro and in vivo in a variety of xenograft tumor models was also evaluated. Finally, the antitumor activity of SGN-B7H4V as monotherapy and in combination with an anti-programmed cell death-1 (PD-1) agent was evaluated using an immunocompetent murine B7-H4-expressing Renca tumor model. RESULTS: Immunohistochemistry confirmed B7-H4 expression across multiple solid tumors, with the highest prevalence in breast, endometrial, and ovarian tumors. In vitro, SGN-B7H4V killed B7-H4-expressing tumor cells by MMAE-mediated direct cytotoxicity and antibody-mediated effector functions including antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. In vivo, SGN-B7H4V demonstrated strong antitumor activity in multiple xenograft models of breast and ovarian cancer, including xenograft tumors with heterogeneous B7-H4 expression, consistent with the ability of vedotin ADCs to elicit a bystander effect. In an immunocompetent murine B7-H4-expressing tumor model, SGN-B7H4V drove robust antitumor activity as a monotherapy that was enhanced when combined with an anti-PD-1 agent. CONCLUSION: The immune checkpoint ligand B7-H4 is a promising molecular target expressed by multiple solid tumors. SGN-B7H4V demonstrates robust antitumor activity in preclinical models through multiple potential mechanisms. Altogether, these preclinical data support the evaluation of SGN-B7H4V as a monotherapy in the ongoing phase 1 study of SGN-B7H4V in advanced solid tumors (NCT05194072) and potential future clinical combinations with immunotherapies.

Stable binding to phosphatidylserine-containing membranes requires conserved arginine residues in tandem C domains of blood coagulation factor VIII.

At sites of vascular damage, factor VIII (fVIII) is proteolytically activated by thrombin and binds to activated platelet surfaces with activated factor IX (fIXa) to form the intrinsic "tenase" complex. Previous structural and mutational studies of fVIII have identified the C1 and C2 domains in binding to negatively charged membrane surfaces through ß-hairpin loops with solvent-exposed hydrophobic residues and a ring of positively charged basic residues. Several hemophilia A-associated mutations within the C domains are suggested to disrupt lipid binding, preventing formation of the intrinsic tenase complex. In this study, we devised a novel platform for generating recombinant C1, C2, and C1C2 domain constructs and performed mutagenesis of several charged residues proximal to the putative membrane binding region of each C domain. Binding measurements between phosphatidylserine (PS)-containing lipid membrane surfaces and fVIII C domains demonstrated an ionic strength dependence on membrane binding affinity. Mutations to basic residues adjacent to the surface-exposed hydrophobic regions of C1 and C2 differentially disrupted membrane binding, with abrogation of binding occurring for mutations to conserved arginine residues in the C1 (R2163) and C2 (R2320) domains. Lastly, we determined the X-ray crystal structure of the porcine fVIII C2 domain bound to o-phospho-L-serine, the polar headgroup of PS, which binds to a basic cleft and makes charge-charge contact with R2320. We conclude that basic clefts in the fVIII C domains bind to PS-containing membranes through conserved arginine residues via a C domain modularity, where each C domain possesses modest electrostatic-dependent affinity and tandem C domains are required for high affinity binding.

An in Vitro Assay Using Cultured Kupffer Cells Can Predict the Impact of Drug Conjugation on in Vivo Antibody Pharmacokinetics.

While antibody-drug conjugates (ADCs) are advancing through clinical testing and receiving new marketing approvals, improvements to the technology continue to be developed in both academic and industrial laboratories. Among the key ADC attributes that can be improved upon with new technology are their biodistribution and pharmacokinetic properties. During the course of ADC development, it has become apparent that conjugation of drugs to the surface of a monoclonal antibody can alter its physicochemical characteristics in a manner that results in increased nonspecific interactions and more rapid elimination from plasma. Researchers in the field have typically relied upon in vivo studies in preclinical models to understand how a particular ADC chemistry will impact these biological characteristics. In previous work, we described how animal studies have revealed a relationship between ADC hydrophobicity, pharmacokinetics, and nonspecific hepatic clearance, particularly by sinusoidal endothelium and Kupffer cells. Here, we describe a fluorescence-based assay using cultured Kupffer cells to recapitulate the nonspecific interactions that lead to ADC clearance in an in vitro setting with the aim of developing a tool for predicting the pharmacokinetics of novel ADC designs. Output from this assay has demonstrated an excellent correlation with plasma clearance for a series of closely related ADCs bearing discrete PEG chains of varying length and has proven useful in interrogating the mechanism of the interactions between ADCs and Kupffer cells.

A coiled-coil masking domain for selective activation of therapeutic antibodies.

The use of monoclonal antibodies in cancer therapy is limited by their cross-reactivity to healthy tissue. Tumor targeting has been improved by generating masked antibodies that are selectively activated in the tumor microenvironment, but each such antibody necessitates a custom design. Here, we present a generalizable approach for masking the binding domains of antibodies with a heterodimeric coiled-coil domain that sterically occludes the complementarity-determining regions. On exposure to tumor-associated proteases, such as matrix metalloproteinases 2 and 9, the coiled-coil peptides are cleaved and antigen binding is restored. We test multiple coiled-coil formats and show that the optimized masking domain is broadly applicable to antibodies of interest. Our approach prevents anti-CD3-associated cytokine release in mice and substantially improves circulation half-life by protecting the antibody from an antigen sink. When applied to antibody-drug conjugates, our masked antibodies are preferentially unmasked at the tumor site and have increased anti-tumor efficacy compared with unmasked antibodies in mouse models of cancer.