Enhancing Fc-mediated effector functions of monoclonal antibodies: The example of HexaBodies.

Since the approval of the CD20-targeting monoclonal antibody (mAb) rituximab for the treatment of lymphoma in 1997, mAb therapy has significantly transformed cancer treatment. With over 90 FDA-approved mAbs for the treatment of various hematological and solid cancers, modern cancer treatment relies heavily on these therapies. The overwhelming success of mAbs as cancer therapeutics is attributed to their broad applicability, high safety profile, and precise targeting of cancer-associated surface antigens. Furthermore, mAbs can induce various anti-tumor cytotoxic effector mechanisms including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), all of which are mediated via their fragment crystallizable (Fc) domain. Over the past decades, these effector mechanisms have been substantially improved through Fc domain engineering. In this review, we will outline the different approaches to enhance Fc effector functions via Fc engineering of mAbs, with a specific emphasis on the so-called "HexaBody" technology, which is designed to enhance the hexamerization of mAbs on the target cell surface, thereby inducing greater complement activation, CDC, and receptor clustering. The review will summarize the development, preclinical, and clinical testing of several HexaBodies designed for the treatment of B-cell malignancies, as well as the potential use of the HexaBody technology beyond Fc-mediated effector functions.

Mutation of the TGN1412 anti-CD28 monoclonal antibody lower hinge confers specific FcγRIIb binding and retention of super-agonist activity.

The agonistic action of several immunomodulatory monoclonal antibodies (mAbs) requires both target antigen binding and clustering of this mAb:target complex by the Fcs interacting with Fcγ receptors (FcγRs), in particular FcγRIIb, on neighboring bystander cells. Fc mutations were made in the immunoglobulin G4 (IgG4)-based TGN1412 anti-CD28 mAb to define the role of FcγR interactions in its "super-agonist" activity. The dual mutation, IgG4-ED269,270 AA, ablated interaction with all human FcγRs and agonistic action was consequentially lost, confirming the FcγR dependence on the action of TGN1412. The IgG4 lower hinge region (F234 L235 G236 G237 ) was modified by L235 E mutation (F234 E235 G236 G237 ), a mutation commonly used to ablate FcγR binding, including in approved therapeutic mAbs. However, rather than ablating all FcγR binding, IgG4-L235 E conferred specific binding to FcγRIIb, the inhibitory Fc receptor. Furthermore, in combination with the core hinge-stabilizing mutation (IgG4-S228 P, L235 E), this mutation increased affinity for FcγRIIb compared with wild-type IgG4. In addition to having FcγRIIb specificity, these engineered TGN1412 antibodies retained their super-agonistic ability, demonstrating that CD28- and FcγRIIb-specific binding are together sufficient for agonistic function. The FcγRIIb-specific nature of IgG4-L235 E has utility for mAb-mediated immune agonism therapies that are dependent on FcγRIIb interaction and of anti-inflammatory mAbs in allergy and autoimmunity that harness FcγRIIb inhibitory signaling.

Improving Antibody Therapeutics by Manipulating the Fc Domain: Immunological and Structural Considerations.

Interactions between the crystallizable fragment (Fc) domain of antibodies and a plethora of cellular Fc receptors (FcRs) or soluble proteins form a critical link between humoral and innate immunity. In particular, the immunoglobulin G Fc domain is critical for the clearance of target cells by processes that include (a) cytotoxicity, phagocytosis, or complement lysis; (b) modulation of inflammation; (c) antigen presentation; (d) antibody-mediated receptor clustering; and (e) cytokine release. More than 30 Fc-engineered antibodies aimed primarily at tailoring these effects for optimal therapeutic outcomes are in clinical evaluation or have already been approved. Nonetheless, our understanding of how FcR engagement impacts various immune cell phenotypes is still largely incomplete. Recent insights into FcR biology coupled with advances in Fc:FcR structural analysis, Fc engineering, and mouse models that recapitulate human biology are helping to fill in existing knowledge gaps. These advances will provide a blueprint on how to fine-tune the Fc domain to achieve optimal therapeutic efficacy.

Margetuximab in HER2-positive metastatic breast cancer.

Several anti-HER2 agents are approved for third-line treatment and beyond (after first-line and second-line); however, no specific treatment strategy is recommended for third-line and beyond. Although these agents improve disease outcomes, HER2-positive metastatic breast cancer remains incurable and there is an unmet need for effective therapies in the later line setting. This review focuses on the development of margetuximab-cmkb, a novel, Fc-engineered, anti-HER2 monoclonal antibody, and its role in the systemic treatment of adult patients with metastatic HER2-positive breast cancer who have received two or more prior anti-HER2 regimens, at least one of which was for metastatic disease.

In about 20% of patients with breast cancer, their tumor cells make too many copies of a protein called HER2. We call them HER2-positive breast cancer cells. HER2 is a protein that signals to breast cancer cells to make them grow. Certain drugs, known as antibodies, are able to bind to the HER2 proteins on the surface of the tumor cells. This stops their signaling and slows down the growth of the tumor cells. These antibodies are called anti-HER2 antibodies. In addition to its 'head' region binding to HER2, the 'tail' region of the anti-HER2 antibody can bind to certain other proteins (receptors) found on the surface of immune cells. When the anti-HER2 antibodies bind to the receptors on immune cells, this starts an anticancer immune response against the HER2-positive breast cancer cells and kills them. This review explains how anti-HER2 antibodies may block and destroy HER2-positive breast cancer cells. In particular, we focus on the beneficial and adverse effects of margetuximab, an anti-HER2 antibody. The tail region of margetuximab has been changed to boost the immune responses against HER2-positive cancer cells. Margetuximab is approved in the USA for patients with HER2-positive metastatic breast cancer after they have already received two or more anti-HER2 therapies. The decision to approve this was based on the pivotal clinical trial SOPHIA.

Development of Therapeutic Antibodies and Modulating the Characteristics of Therapeutic Antibodies to Maximize the Therapeutic Efficacy.

Monoclonal antibodies (mAb) have been used as therapeutic agents for various diseases, and immunoglobulin G (IgG) is mainly used among antibody isotypes due to its structural and functional properties. So far, regardless of the purpose of the therapeutic antibody, wildtype IgG has been mainly used, but recently, the engineered antibodies with various strategies according to the role of the therapeutic antibody have been used to maximize the therapeutic efficacy. In this review paper, first, the overall structural features and functional characteristics of antibody IgG, second, the old and new techniques for antibody discovery, and finally, several antibody engineering strategies for maximizing therapeutic efficacy according to the role of a therapeutic antibody will be introduced.

A robust heterodimeric Fc platform engineered for efficient development of bispecific antibodies of multiple formats.

Bispecific monoclonal antibodies can bind two protein targets simultaneously and enable therapeutic modalities inaccessible by traditional mAbs. Bispecific formats containing a heterodimeric Fc region are of particular interest, as a heterodimeric Fc empowers both bispecificity and altered valencies while retaining the developability and druggability of a monoclonal antibody. We present a robust heterodimeric Fc platform, called the XmAb® bispecific platform, engineered for efficient development of bispecific antibodies and Fc fusions of multiple formats. First, we engineer a purification solution for proteins containing a heterodimeric Fc using engineered isoelectric point differences in the Fc region that enable straightforward purification of the heterodimeric species. Then, we combine this purification solution with a novel set of Fc substitutions capable of achieving heterodimer yields over 95% with little change in thermostability. Next, we illustrate the flexibility of our heterodimeric Fc with a case study in which a wide range of tumor-associated antigen × CD3 bispecifics are generated, differing in choice of tumor antigen, affinities for both tumor antigen and CD3, and tumor antigen valency. Finally, we present manufacturing data reinforcing the robustness of the heterodimeric Fc platform at scale.

Comprehensive elucidation of the structural and functional roles of engineered disulfide bonds in antibody Fc fragment.

Therapeutic monoclonal antibodies and Fc-fusion proteins containing antibody Fc fragment may tend to destabilize (e.g. unfold and aggregate), which leads to loss of functions and increase of adverse risks. Although engineering of an additional disulfide bond has been performed in Fc or Fc domains for optimization, the relationships between introduced disulfide bond and alteration of the stability, aggregation propensity and function were still unclear and should be addressed for achievement of better therapeutic outcome. Here, we constructed three human IgG1 Fc mutants including FcCH2-s-s- (one engineered disulfide bond in CH2 domain), FcCH3-s-s- (one engineered disulfide bond in CH3 domain), and FcCH3-s-s-CH2-s-s- (two engineered disulfide bonds in CH2 and CH3 domains, respectively) for evaluation. As expected, each mutated domain shows obviously increased stability during thermo-induced unfolding, and FcCH3-s-s-CH2-s-s- is most thermo-stable among wildtype Fc (wtFc) and three mutants. The order of overall stability against denaturant is FcCH3-s-s-CH2-s-s- > FcCH2-s-s- > FcCH3-s-s- > wtFc. Then the aggregation propensity was compared among these four proteins. Under conditions of incubation at 60 °C, their aggregation resistance is in the order of FcCH3-s-s-CH2-s-s- > FcCH2-s-s- > FcCH3-s-s- ≈ wtFc. In contrast, the order is FcCH3-s-s-CH2-s-s- > FcCH3-s-s- > FcCH2-s-s- ≈ wtFc under acidic conditions. In addition, the Fc-mediated functions are not obviously affected by engineered disulfide bond. Our results give a comprehensive elucidation of structural and functional effects caused by additional disulfide bonds in the Fc fragment, which is important for Fc engineering toward the desired clinical performance.

Global conformational changes in IgG-Fc upon mutation of the FcRn-binding site are not associated with altered antibody-dependent effector functions.

Antibody engineering is important for many diagnostic and clinical applications of monoclonal antibodies. We recently reported a series of fragment crystallizable (Fc) mutations targeting the neonatal Fc receptor (FcRn) site on a Lewis Y (Ley) binding IgG1, hu3S193. The hu3S193 variants displayed shortened in vivo half-lives and may have potential for radioimaging or radiotherapy of Ley-positive tumors. Here, we report Fc crystal structures of wild-type hu3S193, seven FcRn-binding site variants, and a variant lacking C1q binding or complement-dependent cytotoxicity (CDC) activity. The Fc conformation of the FcRn-binding sites was similar for wild-type and all mutants of hu3S193 Fc, which suggests that FcRn interactions were directly affected by the amino acid substitutions. The C1q-binding site mutant Fc was nearly identical with the wild-type Fc. Surprisingly, several hu3S193 Fc variants showed large changes in global structure compared with wild-type Fc. All hu3S193 Fc mutants had similar antibody-dependent cellular cytotoxicity, despite some with conformations expected to diminish Fc gamma receptor binding. Several hu3S193 variants displayed altered CDC, but there was no correlation with the different Fc conformations. All versions of hu3S193, except the C1q-binding site mutant, bound C1q, suggesting that the altered CDC of some variants could result from different propensities to form IgG hexamers after engaging Ley on target cells. Overall, our findings support the concept that the antibody Fc is both flexible and mobile in solution. Structure-based design approaches should take into account the conformational plasticity of the Fc when engineering antibodies with optimal effector properties.

Myelin oligodendrocyte glycoprotein-specific antibodies from multiple sclerosis patients exacerbate disease in a humanized mouse model.

Myelin oligodendrocyte glycoprotein (MOG) is exposed on the outer surface of the myelin sheath, and as such, represents a possible target antigen for antibodies in multiple sclerosis (MS) and other demyelinating diseases. However, despite extensive analyses, whether MOG-specific antibodies contribute to pathogenesis in human MS remains an area of uncertainty. In the current study we demonstrate that antibodies derived from adult MS patients exacerbate experimental autoimmune encephalomyelitis (EAE) in 'humanized' mice that transgenically express human FcγRs (hFcγRs). Importantly, this exacerbation is dependent on MOG recognition by the human-derived antibodies. The use of mice that express hFcγRs has allowed us to also investigate the contribution of these receptors to disease in the absence of confounding effects of cross-species differences. Specifically, by engineering the Fc region of MOG-specific antibodies to modulate FcγR and complement (C1q) binding, we reveal that FcγRs but not complement activation contribute to EAE pathogenesis. Importantly, selective enhancement of the affinities of these antibodies for specific FcγRs reveals that FcγRIIA is more important than FcγRIIIA in mediating disease exacerbation. These studies not only provide definitive evidence for the contribution of MOG-specific antibodies to MS, but also reveal mechanistic insight that could lead to new therapeutic targets.

An Fc Double-Engineered CD20 Antibody with Enhanced Ability to Trigger Complement-Dependent Cytotoxicity and Antibody-Dependent Cell-Mediated Cytotoxicity.

BACKGROUND: Engineering of the antibody's fragment crystallizable (Fc) by modifying the amino acid sequence (Fc protein engineering) or the glycosylation pattern (Fc glyco-engineering) allows enhancing effector functions of tumor targeting antibodies. Here, we investigated whether complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) of CD20 antibodies could be improved simultaneously by combining Fc protein engineering and glyco-engineering technologies. METHODS AND RESULTS: Four variants of the CD20 antibody rituximab were generated: a native IgG1, a variant carrying the EFTAE modification (S267E/H268F/S324T/G236A/I332E) for enhanced CDC as well as glyco-engineered, non-fucosylated derivatives of both to boost ADCC. The antibodies bound CD20 specifically with similar affinity. Antibodies with EFTAE modification were more efficacious in mediating CDC, irrespective of fucosylation, than antibodies with wild-type sequences due to enhanced C1q binding. In contrast, non-fucosylated variants had an enhanced affinity to FcγRIIIA and improved ADCC activity. Importantly, the double-engineered antibody lacking fucose and carrying the EFTAE modification mediated both CDC and ADCC with higher efficacy than the native CD20 IgG1 antibody. CONCLUSION: Combining glyco-engineering and protein engineering technologies offers the opportunity to simultaneously enhance ADCC and CDC activities of therapeutic antibodies. This approach may represent an attractive strategy to further improve antibody therapy of cancer and deserves further evaluation.

Antigen dynamics govern the induction of CD4(+) T cell tolerance during autoimmunity.

Antigen-specific T cell tolerance holds great promise for the treatment of autoimmune diseases. However, strategies to induce durable tolerance using high doses of soluble antigen have to date been unsuccessful, due to lack of efficacy and the risk of hypersensitivity. In the current study we have overcome these limitations by developing a platform for tolerance induction based on engineering the immunoglobulin Fc region to modulate the dynamic properties of low doses (1 µg/mouse; ∼50 µg/kg) of Fc-antigen fusions. Using this approach, we demonstrate that antigen persistence is a dominant factor governing the elicitation of tolerance in the model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), induced by immunizing B10.PL mice with the N-terminal epitope of myelin basic protein. Unexpectedly, our analyses reveal a stringent threshold of antigen persistence for both prophylactic and therapeutic treatments, although distinct mechanisms lead to tolerance in these two settings. Importantly, the delivery of tolerogenic Fc-antigen fusions during ongoing disease results in the downregulation of T-bet and CD40L combined with amplification of Foxp3(+) T cell numbers. The generation of effective, low dose tolerogens using Fc engineering has potential for the regulation of autoreactive T cells.

Antitumor activities of agonistic anti-TNFR antibodies require differential FcγRIIB coengagement in vivo.

Agonistic anti-TNF receptor (TNFR) superfamily member antibodies are a class of promising antitumor therapies in active clinical investigation. An unexpected requirement for inhibitory Fcγ receptor FcγRIIB coengagement has recently been described for their in vivo antitumor activities. Although these findings have informed the design of more potent antitumor agonistic, anti-TNFR therapies, the underlying mechanism has remained obscure. Through detailed genetic analysis of strains conditionally deleted for FcγRIIB on defined cellular populations or mutated in specific signaling components, we now demonstrate that different agonistic anti-TNFR antibodies have specific requirements for FcγRIIB expression on defined cellular populations and function in trans in the absence of FcγRIIB signaling components, thus supporting a general mechanism of FcγRIIB cross-linking in vivo for the activities of these antibodies.

Low-affinity FcγR interactions can decide the fate of novel human IgG-sensitised red blood cells and platelets.

G1Δnab is a mutant human IgG1 constant region with a lower ability to interact with FcγR than the natural IgG constant regions. Radiolabelled RBCs and platelets sensitised with specific G1Δnab Abs were cleared more slowly from human circulation than IgG1-sensitised counterparts. However, non-destructive splenic retention of G1Δnab-coated RBCs required investigation and plasma radioactivities now suggest this also occurred for platelets sensitised with an IgG1/G1Δnab mixture. In vitro assays with human cells showed that G1Δnab-sensitised RBCs did not cause FcγRI-mediated monocyte activation, FcγRIIIa-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) or macrophage phagocytosis although they did adhere to macrophages. Thus, FcγRII was implicated in the adhesion despite the Δnab mutation reducing the already low-affinity binding to this receptor class. Additional contacts via P-selectin enhance the interaction of sensitised platelets with monocytes and this system provided evidence of FcγRII-dependent activation by G1Δnab. These results emphasise the physiological relevance of low-affinity interactions: It appears that FcγRII interactions of G1Δnab allowed splenic retention of G1Δnab-coated RBCs with inhibitory FcγRIIb binding preventing RBC destruction and that FcγRIIb engagement by G1Δnab on IgG1/G1Δnab-sensitised platelets overcame activation by IgG1. Considering therapeutic blocking Abs, G1Δnab offers lower FcγR binding and a greater bias towards inhibition than IgG2 and IgG4 constant regions.

Protein engineering and the use of molecular modeling and simulation: the case of heterodimeric Fc engineering.

Computational and structure guided methods can make significant contributions to the development of solutions for difficult protein engineering problems, including the optimization of next generation of engineered antibodies. In this paper, we describe a contemporary industrial antibody engineering program, based on hypothesis-driven in silico protein optimization method. The foundational concepts and methods of computational protein engineering are discussed, and an example of a computational modeling and structure-guided protein engineering workflow is provided for the design of best-in-class heterodimeric Fc with high purity and favorable biophysical properties. We present the engineering rationale as well as structural and functional characterization data on these engineered designs.

Fc engineered anti-virus therapeutic human IgG1 expressed in plants with altered binding to the neonatal Fc receptor.

Production of therapeutic monoclonal antibody (mAb) in transgenic plants has several advantages such as large-scale production and the absence of pathogenic animal contaminants. However, mAb with high mannose (HM) type glycans has shown a faster clearance compared to antibodies produced in animal cells. The neonatal Fc receptor (FcRn) regulates the persistence of immunoglobulin G (IgG) by the FcRn-mediated recycling pathway, which salvages IgG from lysosomal degradation within cells. In this study, Fc-engineering of antirabies virus therapeutic mAb SO57 with the endoplasmic reticulum (ER)-retention peptide signal (Lys-Asp-Glu-Leu; KDEL) (mAbpK SO57) in plant cell was conducted to enhance its binding activity to human neonatal Fc receptor (hFcRn), consequently improve its serum half-life. Enzyme-linked immunosorbent assay (ELISA) and Surface plasmon resonance assay showed altered binding affinity of the Fc region of three different mAbpK SO57 variants [M252Y/S254T/T256E (MST), M428L/N434S (MN), H433K/N434F (HN)] to hFcRn compared to wild type (WT) of mAbpK SO57. Molecular modeling data visualized the structural alterations in these mAbpK SO57. All of the mAbpK SO57 variants had HM type glycan structures similar to the WT mAbpK SO57. In addition, the neutralizing activity of the three variants against the rabies virus CVS-11 was effective as the WT mAbpK SO57. These results indicate that the binding affinity of mAbpK SO57 variants to hFcRn can be modified without alteration of N-glycan structure and neutralization activity. Taken together, this study suggests that Fc-engineering of antirabies virus mAb can be applied to enhance the efficacy of therapeutic mAbs in plant expression systems.

Fc engineering by monoclonal mammalian cell display for improved affinity and selectivity towards FcγRs.

Fc optimization can significantly enhance therapeutic efficacy of monoclonal antibodies. However, existing Fc engineering approaches are sub-optimal with noted limitations, such as inappropriate glycosylation, polyclonal libraries, and utilizing fragment but not full-length IgG display. Applying cell cycle arrested recombinase-mediated cassette exchange, this study constructed high-quality monoclonal Fc libraries in CHO cells, displayed full-length IgG on cell surface, and preformed ratiometric fluorescence activated cell sorting (FACS) with the antigen and individual FcγRs. Identified Fc variants were quantitatively evaluated by flow cytometry, ELISA, kinetic and steady-state binding affinity measurements, and cytotoxicity assays. An error-prone Fc library focusing on the hinge-CH2 region was constructed in CHO cells with a functional diversity of 7.5 × 106. Panels of novel Fc variants with enhanced affinity and selectivity for FcγRs were isolated. Particularly, clone 2a-10 (G236E/K288R/K290W/K320M) showed increased binding strength towards FcγRIIa-131R and 131H allotypes with kinetic dissociation constants (KD-K) of 140 nM and 220 nM, respectively, while reduced binding strength towards FcγRIIb compared to WT Fc; clone 2b-1 (K222I/V302E/L328F/K334E) had KD-K of 180 nM towards FcγRIIb; clone 3a-2 (P247L/K248E/K334I) exhibited KD-K of 190 nM and 100 nM towards FcγRIIIa-176F and 176 V allotypes, respectively, and improved potency of 2.0 ng/ml in ADCC assays. Key mutation hotspots were identified, including P247 for FcγRIIIa, K290 for FcγRIIa, and K334 for FcγRIIb bindings. Discovery of Fc variants with enhanced affinity and selectivity towards individual FcγR and the identification of novel mutation hotspots provide valuable insights for further Fc optimization and serve as a foundation for advancing antibody therapeutics development.

Regulatory considerations in the design, development and quality of monoclonal antibodies and related products for the diagnosis and treatment of cancer.

Over 160 therapeutic and in vivo diagnostic monoclonal antibodies have been approved by the US FDA since the first monoclonal antibody, muromonab, was approved in 1986. Approximately 42% of these approvals were for the treatment or in vivo diagnosis of oncology indications, although some products are no longer marketed. This review will look at the history of monoclonal antibody development and approvals, discuss current antibody-based modalities, regulatory considerations for engineering approaches, critical quality attributes for different modalities, immunogenicity of mAbs across oncology products, and the future directions for development of therapeutic and diagnostic monoclonal antibody-based products.

Therapeutic efficacy of a potent anti-Venezuelan equine encephalitis virus antibody is contingent on Fc effector function.

The development of specific, safe, and potent monoclonal antibodies (Abs) has led to novel therapeutic options for infectious disease. In addition to preventing viral infection through neutralization, Abs can clear infected cells and induce immunomodulatory functions through engagement of their crystallizable fragment (Fc) with complement proteins and Fc receptors on immune cells. Little is known about the role of Fc effector functions of neutralizing Abs in the context of encephalitic alphavirus infection. To determine the role of Fc effector function in therapeutic efficacy against Venezuelan equine encephalitis virus (VEEV), we compared the potently neutralizing anti-VEEV human IgG F5 (hF5) Ab with intact Fc function (hF5-WT) or containing the loss of function Fc mutations L234A and L235A (hF5-LALA) in the context of VEEV infection. We observed significantly reduced binding to complement and Fc receptors, as well as differential in vitro kinetics of Fc-mediated cytotoxicity for hF5-LALA compared to hF5-WT. The in vivo efficacy of hF5-LALA was comparable to hF5-WT at -24 and + 24 h post infection, with both Abs providing high levels of protection. However, when hF5-WT and hF5-LALA were administered + 48 h post infection, there was a significant decrease in the therapeutic efficacy of hF5-LALA. Together these results demonstrate that optimal therapeutic Ab treatment of VEEV, and possibly other encephalitic alphaviruses, requires neutralization paired with engagement of immune effectors via the Fc region.

Developability profiling of a panel of Fc engineered SARS-CoV-2 neutralizing antibodies.

To combat the COVID-19 pandemic, potential therapies have been developed and moved into clinical trials at an unprecedented pace. Some of the most promising therapies are neutralizing antibodies against SARS-CoV-2. In order to maximize the therapeutic effectiveness of such neutralizing antibodies, Fc engineering to modulate effector functions and to extend half-life is desirable. However, it is critical that Fc engineering does not negatively impact the developability properties of the antibodies, as these properties play a key role in ensuring rapid development, successful manufacturing, and improved overall chances of clinical success. In this study, we describe the biophysical characterization of a panel of Fc engineered ("TM-YTE") SARS-CoV-2 neutralizing antibodies, the same Fc modifications as those found in AstraZeneca's Evusheld (AZD7442; tixagevimab and cilgavimab), in which the TM modification (L234F/L235E/P331S) reduce binding to FcγR and C1q and the YTE modification (M252Y/S254T/T256E) extends serum half-life. We have previously shown that combining both the TM and YTE Fc modifications can reduce the thermal stability of the CH2 domain and possibly lead to developability challenges. Here we show, using a diverse panel of TM-YTE SARS-CoV-2 neutralizing antibodies, that despite lowering the thermal stability of the Fc CH2 domain, the TM-YTE platform does not have any inherent developability liabilities and shows an in vivo pharmacokinetic profile in human FcRn transgenic mice similar to the well-characterized YTE platform. The TM-YTE is therefore a developable, effector function reduced, half-life extended antibody platform.

Controlled release of enhanced cross-hybrid IgGA Fc PD-L1 inhibitors using oncolytic adenoviruses.

Immune checkpoint inhibitors have clinical success in prolonging the life of many cancer patients. However, only a minority of patients benefit from such therapy, calling for further improvements. Currently, most PD-L1 checkpoint inhibitors in the clinic do not elicit Fc effector mechanisms that would substantially increase their efficacy. To gain potency and circumvent off-target effects, we previously designed an oncolytic adenovirus (Ad-Cab) expressing an Fc fusion peptide against PD-L1 on a cross-hybrid immunoglobulin GA (IgGA) Fc. Ad-Cab elicited antibody effector mechanisms of IgG1 and IgA, which led to higher tumor killing compared with each isotype alone and with clinically approved PD-L1 checkpoint inhibitors. In this study, we further improved the therapy to increase the IgG1 Fc effector mechanisms of the IgGA Fc fusion peptide (Ad-Cab FT) by adding four somatic mutations that increase natural killer (NK) cell activation. Ad-Cab FT was shown to work better at lower concentrations compared with Ad-Cab in vitro and in vivo and to have better tumor- and myeloid-derived suppressor cell killing, likely because of higher NK cell activation. Additionally, the biodistribution of the Fc fusion peptide demonstrated targeted release in the tumor microenvironment with minimal or no leakage to the peripheral blood and organs in mice. These data demonstrate effective and safe use of Ad-Cab FT, bidding for further clinical investigation.