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.

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.

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.

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.

A novel Fc-enhanced humanized monoclonal antibody targeting B7-H3 suppresses the growth of ESCC.

Esophageal squamous cell carcinoma (ESCC) is a prevalent malignant tumor of the digestive tract with a low 5-year survival rate due to the lack of effective treatment methods. Although therapeutic monoclonal antibodies (mAbs) now play an important role in cancer therapy, effective targeted mAbs are still lacking for ESCC. B7-H3 is highly expressed in a variety of tumors and has emerged as a promising therapeutic target. Several mAbs against B7-H3 have advanced to clinical trials, but their development has not yet been pursued for ESCC. Here, we developed a humanized and Fc-engineered anti-B7H3 mAb 24F-Hu-mut2 and systematically evaluated its anti-tumor activity in vitro and in vivo. The 24F-Hu-mut2 was humanized and modified in Fc fragment to obtain stronger antibody-dependent cell-mediated cytotoxicity(ADCC) activity and nanomolar affinity. Furthermore, both of ESCC cell-derived xenograft (CDX) and patient-derived xenograft (PDX) mice models indicated that 24F-Hu-mut2 displayed potent in vivo anti-tumor activity. In addition, a computational docking model showed that the mAb bound to IgC1 and IgC2 domain of B7-H3, which is closer to the cell membrane. Consistently, our ELISA results verified the binding of 24F-Hu-WT and IgC1 and IgC2. Our results indicate that 24F-Hu-mut2 has significant anti-ESCC activity both in vitro and in vivo, and this monoclonal antibody may be a promising antibody against ESCC and other B7-H3 overexpressing tumors.

Fc-Engineered Therapeutic Antibodies: Recent Advances and Future Directions.

Monoclonal therapeutic antibodies have revolutionized the treatment of cancer and other diseases. Fc engineering aims to enhance the effector functions or half-life of therapeutic antibodies by modifying their Fc regions. Recent advances in the Fc engineering of modern therapeutic antibodies can be considered the next generation of antibody therapy. Various strategies are employed, including altering glycosylation patterns via glycoengineering and introducing mutations to the Fc region, thereby enhancing Fc receptor or complement interactions. Further, Fc engineering strategies enable the generation of bispecific IgG-based heterodimeric antibodies. As Fc engineering techniques continue to evolve, an expanding portfolio of Fc-engineered antibodies is advancing through clinical development, with several already approved for medical use. Despite the plethora of Fc-based mutations that have been analyzed in in vitro and in vivo models, we focus here in this review on the relevant Fc engineering strategies of approved therapeutic antibodies to finetune effector functions, to modify half-life and to stabilize asymmetric bispecific IgGs.

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.

Complement Activation by an Anti-Dengue/Zika Antibody with Impaired Fcγ Receptor Binding Provides Strong Efficacy and Abrogates Risk of Antibody-Dependent Enhancement.

To combat infectious diseases, vaccines are considered the best prophylactic strategy for a wide range of the population, but even when vaccines are effective, the administration of therapeutic antibodies against viruses could provide further treatment options, particularly for vulnerable groups whose immunity against the viruses is compromised. Therapeutic antibodies against dengue are ideally engineered to abrogate binding to Fcγ receptors (FcγRs), which can induce antibody-dependent enhancement (ADE). However, the Fc effector functions of neutralizing antibodies against SARS-CoV-2 have recently been reported to improve post-exposure therapy, while they are dispensable when administered as prophylaxis. Hence, in this report, we investigated the influence of Fc engineering on anti-virus efficacy using the anti-dengue/Zika human antibody SIgN-3C and found it affected the viremia clearance efficacy against dengue in a mouse model. Furthermore, we demonstrated that complement activation through antibody binding to C1q could play a role in anti-dengue efficacy. We also generated a novel Fc variant, which displayed the ability for complement activation but showed very low FcγR binding and an undetectable level of the risk of ADE in a cell-based assay. This Fc engineering approach could make effective and safe anti-virus antibodies against dengue, Zika and other viruses.

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.

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.

Outsmarting Pathogens with Antibody Engineering.

There is growing interest in identifying antibodies that protect against infectious diseases, especially for high-risk individuals and pathogens for which no vaccine is yet available. However, pathogens that manifest as opportunistic or latent infections express complex arrays of virulence-associated proteins and are adept at avoiding immune responses. Some pathogens have developed strategies to selectively destroy antibodies, whereas others create decoy epitopes that trick the host immune system into generating antibodies that are at best nonprotective and at worst enhance pathogenesis. Antibody engineering strategies can thwart these efforts by accessing conserved neutralizing epitopes, generating Fc domains that resist capture or degradation and even accessing pathogens hidden inside cells. Design of pathogen-resistant antibodies can enhance protection and guide development of vaccine immunogens against these complex pathogens. Here, we discuss general strategies for design of antibodies resistant to specific pathogen defense mechanisms.

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.

Engineering a pure and stable heterodimeric IgA for the development of multispecific therapeutics.

ABBREVIATIONS: CE-SDS: capillary electrophoresis sodium dodecyl sulfate; DSC: differential scanning calorimetry; FACS: fluorescence-activated cell sorting; FSA: full-sized antibody; Her2: human epidermal growth factor receptor 2; MFI: mean fluorescent intensity; OAA: one-armed antibody; PBS: phosphate-buffered saline; PDB: Protein Data Bank; SEC: size-exclusion chromatography; prepSEC (preparative SEC); RMSD: root-mean-square deviation; RU: resonance units; SPR: surface plasmon resonance; TAA: tumor-associated antigen; WT: wild-type.

Monoclonal Antibodies for Bacterial Pathogens: Mechanisms of Action and Engineering Approaches for Enhanced Effector Functions.

Monoclonal antibody (mAb) therapy has opened a new era in the pharmaceutical field, finding application in various areas of research, from cancer to infectious diseases. The IgG isoform is the most used therapeutic, given its long half-life, high serum abundance, and most importantly, the presence of the Fc domain, which can be easily engineered. In the infectious diseases field, there has been a rising interest in mAbs research to counteract the emerging crisis of antibiotic resistance in bacteria. Various pathogens are acquiring resistance mechanisms, inhibiting any chance of success of antibiotics, and thus may become critically untreatable in the near future. Therefore, mAbs represent a new treatment option which may complement or even replace antibiotics. However, very few antibacterial mAbs have succeeded clinical trials, and until now, only three mAbs have been approved by the FDA. These failures highlight the need of improving the efficacy of mAb therapeutic activity, which can also be achieved with Fc engineering. In the first part of this review, we will describe the mechanisms of action of mAbs against bacteria, while in the second part, we will discuss the recent advances in antibody engineering to increase efficacy of pre-existing anti-bacterial mAbs.

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.

A novel IgG Fc by computer-aided design enhances heavy-chain heterodimerization in bi- or trispecific antibodies.

The classical `knob-into-holes' (KIH) strategy (knob(T366Y)/hole (Y407T)) has successfully enhanced the heterodimerization of a bispecific antibody (BsAb) resulting in heterodimer formation up to 92% of protein A (ProA)-purified protein pool. However, it does not show high efficiency for every BsAb. KIH was initially applied to a CD20/CD3 BsAb. After in silico modeling, two additional new mutations, S354Y in knob-heavy chain (HC) and Q347E in hole-HC, together with KIH named `ETYY', were introduced in the Fc. The CD20/CD3 BsAb hybrid only represented ~ 50% of the ProA-purified protein pool when KIH was applied. With ETYY, the percentage of CD20/CD3 hybrid increased to 93.8%. CD20/CD3-v4b (containing ETYY) retains the original activity of the BsAb at both Fab and Fc regions, and also shows good developability. These results indicate that the computer-aided novel ETYY design has the potential to improve the development of next-generation BsAbs with higher yields and simpler purification.

Full-length recombinant antibodies from Escherichia coli: production, characterization, effector function (Fc) engineering, and clinical evaluation.

Although several antibody fragments and antibody fragment-fusion proteins produced in Escherichia coli (E. coli) are approved as therapeutics for various human diseases, a full-length monoclonal or a bispecific antibody produced in E. coli has not yet been approved. The past decade witnessed substantial progress in expression of full-length antibodies in the E. coli cytoplasm and periplasm, as well as in cell-free expression systems. The equivalency of E. coli-produced aglycosylated antibodies and their mammalian cell-produced counterparts, with respect to biochemical and biophysical properties, including antigen binding, in vitro and in vivo serum stability, pharmacokinetics, and in vivo serum half-life, has been demonstrated. Extensive engineering of the Fc domain of aglycosylated antibodies enables recruitment of various effector functions, despite the lack of N-linked glycans. This review summarizes recent research, preclinical advancements, and clinical development of E. coli-produced aglycosylated therapeutic antibodies as monoclonal, bispecific, and antibody-drug conjugates for use in autoimmune, oncology, and immuno-oncology areas.Abbreviations: ADA Anti-drug antibody; ADCC Antibody-dependent cellular cytotoxicity; ADCP Antibody-dependent cellular phagocytosis; ADC Antibody-drug conjugate; aFc Aglycosylated Fc; AMD Age-related macular degeneration aTTP Acquired thrombotic thrombocytopenic purpura; BCMA B-cell maturation antigen; BLA Biologics license application; BsAb Bispecific antibody; C1q Complement protein C1q; CDC Complement-dependent cytotoxicity; CDCC Complement-dependent cellular cytotoxicity; CDCP Complement-dependent cellular phagocytosis; CEX Cation exchange chromatography; CFPS Cell-free protein expression; CHO Chinese Hamster Ovary; CH1-3 Constant heavy chain 1-3; CL Constant light chain; DLBCL Diffuse large B-cell lymphoma; DAR Drug antibody ratio; DC Dendritic cell; dsFv Disulfide-stabilized Fv; EU European Union; EGFR Epidermal growth factor receptor; E. coli Escherichia coli; EpCAM Epithelial cell adhesion molecule; Fab Fragment antigen binding; FACS Fluorescence activated cell sorting; Fc Fragment crystallizable; FcRn Neonatal Fc receptor; FcÉ£Rs Fc gamma receptors; FDA Food and Drug Administration; FL-IgG Full-length immunoglobulin; Fv Fragment variable; FolRαa Folate receptor alpha; gFc Glycosylated Fc; GM-CSF Granulocyte macrophage-colony stimulating factor; GPx7 Human peroxidase 7; HCL Hairy cell leukemia; HIV Human immunodeficiency virusl; HER2 Human epidermal growth factor receptor 2; HGF Hepatocyte growth factor; HIC Hydrophobic interaction chromatography; HLA Human leukocyte antigen; IBs Inclusion bodies; IgG1-4 Immunoglobulin 1-4; IP Intraperitoneal; ITC Isothermal titration calorimetry; ITP Immune thrombocytopenia; IV Intravenous; kDa Kilodalton; KiH Knob-into-Hole; mAb Monoclonal antibody; MAC Membrane-attack complex; mCRC Metastatic colorectal cancer; MM Multipl myeloma; MOA Mechanism of action; MS Mass spectrometry; MUC1 Mucin 1; MG Myasthenia gravis; NB Nanobody; NK Natural killer; nsAA Nonstandard amino acid; NSCLC Non-small cell lung cancer; P. aeruginosa Pseudomonas aeruginosa; PD-1 Programmed cell death 1; PD-L1 Programmed cell death-ligand 1; PDI Protein disulfide isomerase; PECS Periplasmic expression cytometric screening; PK Pharmacokinetics; P. pastoris Pichia pastoris; PTM Post-translational modification; Rg Radius of gyration; RA Rheumatoid arthritis; RT-PCR Reverse transcription polymerase chain reaction; SAXS Small angle X-ray scattering; scF Single chain Fv; SCLC Small cell lung cancer; SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEC Size exclusion chromatography; SEED Strand-exchange engineered domain; sRNA Small regulatory RNA; SRP Signal recognition particle; T1/2 Half-life; Tagg Aggregation temperature; TCR T cell receptor; TDB T cell-dependent bispecific; TF Tissue factor; TIR Translation initiation region; Tm Melting temperature; TNBC Triple-negative breast cancer; TNF Tumor necrosis factor; TPO Thrombopoietin; VEGF Vascular endothelial growth factor; vH Variable heavy chain; vL Variable light chain; vWF von Willebrand factor; WT Wild type.

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

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

Effect of the ADCC-Modulating Mutations and the Selection of Human IgG Isotypes on Physicochemical Properties of Fc.

Monoclonal antibodies, particularly IgGs and Ig-based molecules, are a well-established and growing class of biotherapeutic drugs. In order to improve efficacy, potency and pharmacokinetics of these therapeutic drugs, pharmaceutical industries have investigated significantly in engineering fragment crystallizable (Fc) domain of these drugs to optimize the interactions of these drugs and Fc gamma receptors (FcγRs) in recent ten years. The biological function of the therapeutics with the antibody-dependent cellular cytotoxicity (ADCC) enhanced double mutation (S239D/I332E) of isotype IgG1, the ADCC reduced double mutation (L234A/L235A) of isotype IgG1, and ADCC reduced isotype IgG4 has been well understood. However, limited information regarding the effect of these mutations or isotype difference on physicochemical properties (PCP), developability, and manufacturability of therapeutics bearing these different Fc regions is available. In this report, we systematically characterize the effects of the mutations and IgG4 isotype on conformation stability, colloidal stability, solubility, and storage stability at accelerated conditions in two buffer systems using six Fc variants. Our results provide a basis for selecting appropriate Fc region during development of IgG or Ig-based therapeutics and predicting effect of the mutations on CMC development process.