Fc Interactions Research Articles

Impact of site-specific conjugation strategies on the pharmacokinetics of antibody conjugated radiotherapeutics

Antibody radionuclide conjugates are an emerging modality for targeted imaging and potent therapy of disseminated disease. Coupling of radionuclides to monoclonal antibodies (mAbs) is typically achieved by applying non-site-specific labelling techniques. With the ambition of reducing variability, increasing labelling efficacy and stability, several site-specific conjugation strategies have been developed in recent years for toxin- and fluorophore-mAb conjugates. In this study, we studied two site-specific labelling strategies for the conjugation of the macrocyclic chelating agent, DOTA, to the anti-Leucine Rich Repeat Containing 15 (LRRC15) mAb DUNP19. Specifically, one approach utilized a DOTA-bearing peptide (FcIII) with a strong affinity for the fragment crystallizable (Fc) domain of the human IgG1 of DUNP19 (DUNP19LF-FcIII-DOTASS), while the other leveraged a chemo-enzymatic technique to substitute the N-linked bi-antennary oligosaccharides in the human IgG1 Fc domain with DOTA (DUNP19LF-gly-DOTASS). To assess if these methods impact the antibody's binding properties and targeting efficacy, comparative in vitro and in vivo studies of the generated DUNP19-conjugates were performed. While the LRRC15 binding of both radioimmunoconjugates remained intact, the conjugation methods had different impacts on their abilities to interact with FcRn and FcγRs. In vitro assessments of DUNP19LF-FcIII-DOTASS and DUNP19LF-gly-DOTASS demonstrated markedly decreased affinity for FcRn and FcγRIIIa (CD16), respectively. DUNP19LF-FcIII-DOTASS demonstrated increased blood and tissue kinetics in vivo, confirming loss of FcRn binding. While the ablated FcγR interaction of DUNP19LF-gly-DOTASS had no immediate impact on in vivo biodistribution, reduced immunotherapeutic effect can be expected in future studies as a result of reduced NK-cells interaction. In conclusion, our findings underscore the necessity for meticulous consideration and evaluation of mAb labelling strategies, extending beyond mere conjugation efficiency and radiolabeling yields. Notably, site-specific labelling methods were found to significantly influence the immunological impact of Fc interactions. Therefore, it is of paramount importance to consider the intended diagnostic or therapeutic application of the construct and to adopt conjugation strategies that ensure the preservation of critical pharmacological properties and functionality of the antibody in use.

Engineered Antibodies as Cancer Radiotheranostics.

Radiotheranostics is a rapidly growing approach in personalized medicine, merging diagnostic imaging and targeted radiotherapy to allow for the precise detection and treatment of diseases, notably cancer. Radiolabeled antibodies have become indispensable tools in the field of cancer theranostics due to their high specificity and affinity for cancer-associated antigens, which allows for accurate targeting with minimal impact on surrounding healthy tissues, enhancing therapeutic efficacy while reducing side effects, immune-modulating ability, and versatility and flexibility in engineering and conjugation. However, there are inherent limitations in using antibodies as a platform for radiopharmaceuticals due to their natural activities within the immune system, large size preventing effective tumor penetration, and relatively long half-life with concerns for prolonged radioactivity exposure. Antibody engineering can solve these challenges while preserving the many advantages of the immunoglobulin framework. In this review, the goal is to give a general overview of antibody engineering and design for tumor radiotheranostics. Particularly, the four ways that antibody engineering is applied to enhance radioimmunoconjugates: pharmacokinetics optimization, site-specific bioconjugation, modulation of Fc interactions, and bispecific construct creation are discussed. The radionuclide choices for designed antibody radionuclide conjugates and conjugation techniques and future directions for antibody radionuclide conjugate innovation and advancement are also discussed.

Affinity-Resolved Size Exclusion Chromatography Coupled to Mass Spectrometry: A Novel Tool to Study the Attribute-and-Function Relationship in Therapeutic Monoclonal Antibodies.

Assessment of critical quality attributes (CQAs) is an important aspect during the development of therapeutic monoclonal antibodies (mAbs). Attributes that affect either the target binding or Fc receptor engagement may have direct impacts on the drug safety and efficacy and thus are considered as CQAs. Native size exclusion chromatography (SEC)-based competitive binding assay has recently been reported and demonstrated significant benefits compared to conventional approaches for CQA identification, owing to its faster turn-around and higher multiplexity. Expanding on the similar concept, we report the development of a novel affinity-resolved size exclusion chromatography-mass spectrometry (AR-SEC-MS) method for rapid CQA evaluation in therapeutic mAbs. This method features wide applicability, fast turn-around, high multiplexity, and easy implementation. Using the well-studied Fc gamma receptor III-A (FcγRIIIa) and Fc interaction as a model system, the effectiveness of this method in studying the attribute-and-function relationship was demonstrated. Further, two case studies were detailed to showcase the application of this method in assessing CQAs related to antibody target binding, which included unusual N-linked glycosylation in a bispecific antibody and Met oxidation in a monospecific antibody, both occurring within the complementarity-determining regions (CDRs).

Fc–Fc interactions and immune inhibitory effects of IgG4: implications for anti-PD-1 immunotherapies

BackgroundThe majority of anti-programmed cell-death 1 (PD-1) monoclonal antibodies (mAbs) use S228P mutation IgG4 as the structural basis to avoid the activation of immune cells or complement. However, little attention...

P166 Optimizing PD-1 agonist signaling with membrane proximal binding of rosnilimab, a clinical stage PD-1 agonist IgG1 antibody

Abstract Background Checkpoint agonism represents a promising class of therapies for the treatment of autoimmune and inflammatory diseases, including ulcerative colitis (UC), where unmet needs persist despite available therapies. Binding to membrane proximal regions of checkpoint receptors, together with Fc interactions with receptors on opposing cells, can contribute to tight immune synapse formation between the immune cell and antigen presenting cell. This has been proposed to improve potency of agonistic signaling by excluding phosphatases such as CD45 from the immune synapse and promoting receptor clustering. Optimization of these characteristics results in improved agonism and depletion, with the potential for restoration of immune balance and broader clinical outcomes. Rosnilimab was engineered to leverage these important characteristics. It is a PD-1 agonist IgG1 antibody designed to optimize inhibitory signaling through the PD-1 receptor and to deplete PD-1high pathogenic T cells. Rosnilimab is in Phase 2 clinical development for UC and rheumatoid arthritis. Methods Mutations to surface exposed regions of the PD-1 extracellular domain were made and surface plasmon resonance was used to infer the epitopes of PD-1 agonist molecules from resulting changes to binding. Membrane proximal and distal binding antibodies were studied in in vitro functional assays to assess T cell proliferation and antibody-dependent cellular toxicity. Results Epitopes of agonistic antibodies were mapped to locations on the PD-1 receptor. The membrane proximal binding epitope of rosnilimab was confirmed and binding epitopes for other reference antibodies (ref) were identified. Rosnilimab and a membrane distally binding antibody (ref 1) were selected for comparison in functional assays. Rosnilimab demonstrated greater reduction of T cell proliferation and depletion of PD-1+ T cells compared to ref 1, consistent with the hypothesis that membrane proximal binding improves agonistic activity and target cell depletion. Conclusion By targeting and leveraging inhibitory immune regulatory mechanisms to modulate the pathogenic T cells driving disease, there is an opportunity to dampen the inflammatory cycle and restore immune balance via agonism. Rosnilimab binds to a membrane proximal region of the PD-1 receptor, resulting in potent reduction of T cell proliferation and depletion of PD-1high T cells. These mechanistic data, translational in vivo and in vitro data, robust Phase 1 healthy volunteer data, and unmet needs in UC provide rationale for an ongoing global Phase 2 study of rosnilimab in UC (NCT06127043).

Electrochemical sensing of bisphenol A based on carboxylated multiwalled carbon nanotubes-ferrocene-H5PMo10V2O40 with chitosan for signal amplification

A synergetic signal amplification detection platform based on carboxylated multiwalled carbon nanotubes-ferrocene-H5PMo10V2O40 (CMWNT-Fc-PMo10V2) composite is initially developed to detect bisphenol A (BPA). The CMWNT-Fc-PMo10V2 composite has been synthesized by the interaction of Fc and PMo10V2 with CMWNT as the loading support. Furthermore, the CMWNT-Fc-PMo10V2 composite with negative charge could be firmly immobilized on the cationic chitosan (CHIT) matrix with film-forming ability through the electrostatic attraction, which could overcome the problems that Fc and PMo10V2 could not be stably fixed on the electrode surface. Benefiting from the large surface area and good conductivity of CMWNT, fast electron-transfer rate and low oxidation potential of Fc, and strong redox property of PMo10V2, CMWNT-Fc-PMo10V2-based sensor demonstrates synergistic effect in electrochemical detection of BPA. Under the optimal experimental conditions, the wide linear range from 0.06 to 100 μM, and low detection limit of 0.035 μM for BPA was achieved at the CMWNT-Fc-PMo10V2 sensing interface. Moreover, the newly developed electrochemical method was successfully used for determination of BPA in milk samples with satisfactory results.

Associations between the Big Five personality traits, testosterone, and cortisol in adolescent male athletes.

Testosterone (T) has been conceptualized as a biomarker of individual differences, yet T associations with the Big Five personality traits are inconsistent. Athletes provide a suitable model for evaluation here, as T co-expresses traits related to male-to-male competition and fitness with cortisol (C) playing a moderating role. This study investigated associations between the Big Five traits, T, and C in adolescent male athletes. One hundred and twenty male ice hockey players (aged 14-19 years) were assessed for blood total (T, C) and free (FT, FC) hormones, body-size dimensions (i.e., body mass, height, body mass index [BMI]), the Big Five personality traits (i.e., extraversion, neuroticism, agreeableness, conscientiousness, openness), and trait anxiety. Correlational and regression (with age and BMI as covariates) analyses identified a positive effect of FT on extraversion, but a negative FT effect on neuroticism and anxiety (p < 0.05). Significant FT × FC interactions emerged for extraversion and agreeableness. Slope testing revealed that FT had a positive effect on extraversion at the FC mean and +1 SD, and a negative effect on agreeableness with FC at +1 SD. In conclusion, adolescent male athletes with a higher serum FT concentration tended to express higher extraversion, but lower neuroticism and anxiety. The FT association with extraversion was moderated by FC concentration, as was agreeableness. Therefore, high-FT athletes presented a behavioural disposition that favours dominance and resiliency, with some dependencies on FC availability. Since all association effect sizes were weak, replicate studies on larger adolescent samples are needed.

A novel trivalent non-Fc anti-CD3 Collabody preferentially induces Th1 cell apoptosis in vitro and long-lasting remission in recent-onset diabetic NOD mice.

Specific anti-CD3 treatment is deemed to be a promising therapy for allograft rejection and type 1 diabetes (T1D). Fc receptor (FcR) reduced-binding antibodies, by avoiding adverse effects of Fc and FcR interaction, have good therapeutic potential. We generated a trivalent anti-mouse-CD3 Collabody, h145CSA, by using a triplex-forming collagen-like peptide (Gly-Pro-Pro)10 to drive the trimerization of the Fab fragments. Exposure to h145CSA, but not its bivalent counterparts 145-2C11 and h145chIgGAA (FcR reduced-binding format), upregulates FasL expression on Th1 cells and causes Th1 cell apoptosis. Administration of h145CSA invokes minimal mitogenic effects in mice. The ability of multiple dosing of h145CSA to induce splenic CD4+ T-cell depletion is comparable to bivalent antibodies but is characterized by more rapid CD4+ T-cell recovery kinetics. h145CSA is more potent than h145chIgGAA in inducing long-lasting remission in recent-onset diabetic NOD mice. Its therapeutic effect is accompanied by a significantly lower percentage of CD4+IFNγ+ T cells and a higher Treg/Th1 ratio in pancreatic and mesenteric lymph nodes. The results of our study demonstrate that trivalent non-Fc anti-CD3 Collabody has the potential to be used in the treatment of T1D.

Splitting the reward: Differences in inflammatory marker associations with neural connectivity between reward anticipation and reward outcome in adolescents at high risk for depression

BackgroundAdolescent depression is associated with both dysfunction in neural reward processing and peripheral inflammatory markers (PIMs), such as interleukin-6 (IL-6), C-reactive-protein (CRP), and tumor-necrosis factor alpha (TNFα). Few adolescent studies have examined neural-inflammatory marker associations and associated behavioral correlates, which would contribute to a better understanding of developmental processes linked to depression. Methods36 adolescents at high risk of depression completed an fMRI reward task (during anticipation and outcome), blood draw for PIMs (IL-6, CRP, and TNFα), and a behavioral task assessing motivation to expend effort. Analyses examined associations of task-dependent functional connectivity (FC; ventral striatum to frontal and default mode network brain regions), and if the interaction of PIMs and task-dependent FC predicted motivation to expend effort. ResultsFor anticipation contrast, TNFα was associated with increased task-dependent FC between the LVS and PCC/vmPFC. In moderation analyses, for anticipation contrasts, the combination of higher IL-6 and stronger FC (LVS-precuneus/PCC) was associated with lower motivation to expend effort, while for outcome contrasts, the combination of higher IL-6 and stronger FC (VS-precuneus/PCC) was associated with greater motivation to expend effort. ConclusionsOur findings in adolescents during an important developmental time period suggest that PIMs are directly linked to greater FC between the VS and DMN brain regions during win anticipation, consistent with prior studies. Effects of PIMs on motivation to expend effort may vary the strength/type of neural reward processing (anticipation or outcome), which could guide better understanding how inflammatory markers and neural reward substrates contribute to development of depression in high-risk adolescents.

Novel Sirpα-Fc Fusion Protein IMM01 Exhibits Dual Anti-Tumor Activities By Targeting CD47/Sirpα Signal Pathway

Background: This study aims to evaluate the anti-tumor mechanism of IMM01, a novel recombinant SIRPα-Fc fusion protein targeting CD47-SIRPα pathway. Methods: IMM01 was a recombinant fusion protein of SIRPα (V2) extracellular segment domain 1 and human IgG1 Fc, with the N glycosylation site N80 mutated to A (Alanine) in D1 region. IMM01 fusion proteins were produced using in house developed CHO-K1 cell expression system. IMM01 blocking activities on Jurkat-CSR (Jurkat-CD47KO-CARSIRPα) cells, antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays were done by flow cytometry. Detection of cytokines of IL-1β, IL-2, IL-4, IL-5, IL-6, GM-CSF, IFN-γ, IL-10 and TNF production by PBMC was done CBA assay. In vivo mechanism of action study was done in different mouse models including HL-60 CB17-SCID, Daudi CB17-SCID, SNU-1 CB17-SCID, Raji CB17-SCID and CT26-hPDL1(Tg) hCD47(Tg) mPDL1(KO) mCD47(KO). In addition, human red blood cells binding and erythrocyte agglutination of IMM01 were also evaluated. Results:In vitro binding assay revealed that IMM01 has a strong binding affinity to CD47 with lowest EC50 at 0.4967 nM. The removal of N-glycosylation modification improves the consistence and purity of the IMM01 protein expression, and the potential mitigation of immunogenicity. Using a chimeric SIRPα receptor protein expressing cell line, IMM01 not only can block the CD47 at the protein level, but also block the signal transduction at the cellular levels. Compared to negative (hIgG1-Fc) controls, IMM01 stimulated secretion of minimal amount of IL-10 and TNF but inhibited IL-8 production by PBMC. IMM01 can induce strong ADCP and moderate ADCC, but not the CDC activities. The strong phagocytosis against tumor cells is dependent on interaction of Fc with activating FcγRs on the presence of macrophages. Mechanism of action studies demonstrated that IMM01 induced strong phagocytosis against tumor cells was attributed to dual activities of blocking "don't eat me" signal and activating "eat me" signal. IMM01 has strong and robust in vivo anti-tumor activities in different mouse tumor models either as monotherapy on hematological malignancies, or in combination therapy with PD-L1 mAb, PD-1 mAb, CD33 mAb, and HER-2 mAb, respectively. The in vivo therapeutic efficacy of IMM01 was done in HL-60 xenograft model showed that 100% of the mice achieved CR after administration of IMM01 at 5mg/kg for 2 weeks. Also, 100% of mice achieved CR after IMM01 combined with the above mentioned mAbs in different mouse models, respectively. This illustrated that IMM01 has a great therapeutic potential in combination with these commonly used immunotherapy or targeted therapy agents to be used in the treatment of solid tumors and hematological malignancies. Finally, IMM01 demonstrated a favorable safety profile with no human RBC binding, nor hemagglutination induction. Conclusion: Our study results demonstrated that, as a novel bi-functional SIRPα-Fc recombinant protein, IMM01 has very strong anti-tumor activities with a good curative effect on mouse xenograft tumor models with dual anti-tumor activities by blocking CD47 "don't eat me" signal and activating the phagocytosis "eat me" signal. Furthermore, IMM01 demonstrated a well synergistic effect when combined with different immunotherapeutic agents. Importantly, our results revealed that IMM01 has a favorable safety profile with no human RBC binding activity and no hemagglutination induction. Phase I clinical study is ongoing, and the clinical trial results will be reported soon. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Antibody Engineering for Nuclear Imaging and Radioimmunotherapy.

Radiolabeled antibodies have become indispensable tools in nuclear medicine. However, the natural roles of antibodies within the immune system mean that they have several intrinsic limitations as a platform for radiopharmaceuticals. In recent years, the field has increasingly turned to antibody engineering to circumvent these issues while retaining the manifold benefits of the immunoglobulin framework. In this "Focus on Molecular Imaging" review, we cover recent advances in the application of antibody engineering to immunoPET, immunoSPECT, and radioimmunotherapy. Specifically, we address how antibody engineering has been used to improve radioimmunoconjugates on four fronts: optimizing pharmacokinetics, facilitating site-specific bioconjugation, modulating Fc interactions, and creating bispecific constructs.

Si-containing 3D cage-functionalized graphene oxide grafted with Ferrocene for high-performance supercapacitor application: An experimental and theoretical study

In this work, graphene oxide sheets were functionalized with Octa(aminopropyl)silsesquioxane. Then, Octa(aminopropyl)silsesquioxane-functionalized graphene oxide (GO-Amine-SSQ) was grafted with Ferrocene through Friedel-Craft reaction. Structural properties of the prepared composite (GO-Amine-SSQ-Fc) were analyzed by XPS, FT-IR, XRD, Raman, SEM, TEM, and BET tests. Results confirmed the successful synthesis and high porosity. Next, the electrochemical properties of GO-Amine-SSQ-Fc were characterized by CV, GCD, and EIS techniques in the 3E system. The GO-Amine-SSQ-Fc electrode showed a specific capacitance of 574 F g−1 at 1 A g−1, retention capacitance of 90.1% after 10,000 charge-discharge cycles, low resistance, and efficient diffusion of ions. After confirming the excellent electrochemical performance of this electrode, a symmetric supercapacitor system (GO-Amine-SSQ-Fc//GO-Amine-SSQ-Fc) was tested by CV and GCD techniques, to determine practical application of system. GO-Amine-SSQ-Fc//GO- Amine-SSQ-Fc system recorded a specific capacitance of 304 F g−1 at 0.5 A g−1, retention capacitance of 92.5% over 10,000 charge-discharge cycles, and specific energy of 10.14 Wh kg−1 at a specific power of 500 W kg−1. Also, the results of computational methodology show that the interaction of SSQ, Fc and GO layer in GO-Amine-SSQ-Fc composite, makes it effective as an electrode material for supercapacitors. This excellent performance, as a result of the unique structure of Amine-SSQ groups and the superior electrochemical behavior of Ferrocene groups, suggests that GO-Amine-SSQ-Fc composite has great potential for energy storage devices.

Fc Binding by FcγRIIa Is Essential for Cellular Activation by the Anti-FcγRIIa mAbs 8.26 and 8.2.

FcγR activity underpins the role of antibodies in both protective immunity and auto-immunity and importantly, the therapeutic activity of many monoclonal antibody therapies. Some monoclonal anti-FcγR antibodies activate their receptors, but the properties required for cell activation are not well defined. Here we examined activation of the most widely expressed human FcγR; FcγRIIa, by two non-blocking, mAbs, 8.26 and 8.2. Crosslinking of FcγRIIa by the mAb F(ab’)2 regions alone was insufficient for activation, indicating activation also required receptor engagement by the Fc region. Similarly, when mutant receptors were inactivated in the Fc binding site, so that intact mAb was only able to engage receptors via its two Fab regions, again activation did not occur. Mutation of FcγRIIa in the epitope recognized by the agonist mAbs, completely abrogated the activity of mAb 8.26, but mAb 8.2 activity was only partially inhibited indicating differences in receptor recognition by these mAbs. FcγRIIa inactivated in the Fc binding site was next co-expressed with the FcγRIIa mutated in the epitope recognized by the Fab so that each mAb 8.26 molecule can contribute only three interactions, each with separate receptors, one via the Fc and two via the Fab regions. When the Fab and Fc binding were thus segregated onto different receptor molecules receptor activation by intact mAb did not occur. Thus, receptor activation requires mAb 8.26 Fab and Fc interaction simultaneously with the same receptor molecules. Establishing the molecular nature of FcγR engagement required for cell activation may inform the optimal design of therapeutic mAbs.

Fc Galactosylation Promotes Hexamerization of Human IgG1, Leading to Enhanced Classical Complement Activation.

Human IgG contains one evolutionarily conserved N-linked glycan in its Fc region at position 297. This glycan is crucial for Fc-mediated functions, including its induction of the classical complement cascade. This is induced after target recognition through the IgG-Fab regions, allowing neighboring IgG-Fc tails to associate through Fc:Fc interaction, ultimately leading to hexamer formation. This hexamerization seems crucial for IgG to enable efficient interaction with the globular heads of the first complement component C1q and subsequent complement activation. In this study, we show that galactose incorporated in the IgG1-Fc enhances C1q binding, C4, C3 deposition, and complement-dependent cellular cytotoxicity in human erythrocytes and Raji cells. IgG1-Fc sialylation slightly enhanced binding of C1q, but had little effect on downstream complement activation. Using various mutations that decrease or increase hexamerization capacity of IgG1, we show that IgG1-Fc galactosylation has no intrinsic effect on C1q binding to IgG1, but enhances IgG1 hexamerization potential and, thereby, complement activation. These data suggest that the therapeutic potential of Abs can be amplified without introducing immunogenic mutations, by relatively simple glycoengineering.

On-target IgG hexamerisation driven by a C-terminal IgM tail-piece fusion variant confers augmented complement activation

The majority of depleting monoclonal antibody (mAb) drugs elicit responses via Fc-FcγR and Fc-C1q interactions. Optimal C1q interaction is achieved through hexameric Fc:Fc interactions at the target cell surface. Herein is described an approach to exploit the tailpiece of the naturally multimeric IgM to augment hexamerisation of IgG. Fusion of the C-terminal tailpiece of IgM promoted spontaneous hIgG hexamer formation, resulting in enhanced C1q recruitment and complement-dependent cytotoxicity (CDC) but with off-target complement activation and reduced in-vivo efficacy. Mutation of the penultimate tailpiece cysteine to serine (C575S) ablated spontaneous hexamer formation, but facilitated reversible hexamer formation after concentration in solution. C575S mutant tailpiece antibodies displayed increased complement activity only after target binding, in-line with the concept of ‘on-target hexamerisation’, whilst retaining efficient in-vivo efficacy and augmented target cell killing in the lymph node. Hence, C575S-tailpiece technology represents an alternative format for promoting on-target hexamerisation and enhanced CDC.

Antibody-Dependent Enhancement (ADE) of SARS-CoV-2 Infection Requires FcγRIIB and Virus-Antibody Complex With Bivalent Interaction

Antibody-dependent enhancement (ADE) has been reported for SARS-CoV and MERS-CoV, suggesting the risk of ADE for antibody-based SARS-CoV-2 vaccines and therapeutics. Understanding the underlying molecular mechanisms behind ADE of SARS-CoV-2 is critical for development of safe and effective therapies. Here, we report that two neutralizing mAbs MW01 and MW05 could enhance the infection of SARS-CoV-2 on FcγRIIB-expressing B cells. Enhancing the interaction of Fc with FcγRIIB could increase the ADE activity mediated by MW01 and MW05. Expression of other FcγRs, such as FcγRIA and FcγRIIA, could decrease ADE activity of these two mAbs by competing with FcγRIIB. X-ray crystal structure determination and S trimer-binding modeling showed that MW01 and MW05 can bind to RBDs in S trimer with both “up” and “down” states. While, the neutralizing mAb MW07, which has no ADE activity only binds to RBD in S trimer with “up” state. Monovalent MW01 and MW05 completely diminished the ADE activity compared with their bivalent counterparts. Moreover, both macropinocytosis and endocytosis are confirmed involving in ADE of SARS-CoV-2 infection. Blocking endosome transportation and lysosome acidification could inhibit the ADE activity mediated by MW05. Together, our results identified a novel ADE mechanism of SARS-CoV-2, FcγRIIB-mediated uptake of SARS-CoV-2/mAb complex with bivalent interaction. These findings provide a novel perspective on the mechanism of ADE of SARS-CoV-2, which is informative to the development of safer therapeutics and vaccines. Funding Information: This work was supported by National Key R&D Program (2020YFC0848600 to S.W. and 2020YFC0860700 to L.W.). Declaration of Interests: X.G., S.W., S.J., W.J. and C.G. are listed as inventors on the licensed patents for MW05 and MW07. S.W., S.J., W.J., M.W., Z.L., C.G., B.C., H.C., A.W., G.L., C.G., X.G., J.Z. and D.L. are employees of Mabwell (Shanghai) Bioscience Co., Ltd. and may hold shares in Mabwell (Shanghai) Bioscience Co., Ltd. The other authors declare no competing interests.

Abstract 953: Efficacy of amivantamab, a bispecific EGFR/MET antibody, correlates with EGFR expression and signaling in NSCLC models with wild-type EGFR

Abstract Small molecule EGFR inhibitors have proven effective in treatment of Non-Small Cell Lung Cancer (NSCLC) with activating EGFR mutations, however there is minimal to no efficacy in wild-type EGFR NSCLC. Amivantamab, an EGFR/MET bispecific antibody, has demonstrated clinical activity across a diverse range of EGFR activating mutations in an ongoing Phase I trial and differentiates from anti-EGFR antibodies tested previously in NSCLC clinical trials due to dual targeting and enhanced interaction with Fc receptors on immune cells. As candidate biomarkers to predict response in wt EGFR disease, EGFR and MET expression (immunohistochemistry (IHC)) and signaling (proximity ligation assays (PLA)), as well as tumor associated macrophage (TAM) content, were assessed in 39 NSCLC wt EGFR patient-derived xenograft (PDX) models. A strong correlation (EGFR r=0.63; p&amp;lt;0.0001; MET r=0.83; p&amp;lt;0.0001) between the IHC and PLA scores for both EGFR and MET confirmed the expected link between signaling level and receptor expression. Based on these data, 14 models with different levels of EGFR and MET expression and TAM infiltration were selected for amivantamab efficacy assessment. While no correlation was found between MET IHC H-score and efficacy, positive correlations were identified between EGFR H-score and PLA-score to amivantamab efficacy. Exclusion of models with known oncogenic driver mutations downstream of EGFR and MET, such as KRAS and PI3K, led to strong correlations to both EGFR expression and signaling (IHC - r=0.90; p=0.002; PLA - r=0.92; p=0.001). Indeed, models with EGFR H-score ≥170 and no alternative driver mutations achieved prolonged tumor stasis or complete tumor regressions with amivantamab treatment. No correlation between TAM content and amivantamab efficacy was observed. Previous studies have demonstrated superior amivantamab efficacy in models driven by either mutant EGFR or amplification of MET, compared to a version lacking Fc interaction; however, both antibodies demonstrated similar efficacy in 13 out of 14 NSCLC wt EGFR PDX models. This suggests that while amivantamab efficacy requires Fc-interaction in models driven by EGFR mutation or MET amplification (ligand-independent), the Fc-interaction is not required for efficacy in wt EGFR driven NSCLC models. We hypothesize that NSCLC PDX models with higher levels of wt EGFR are more growth dependent on EGFR ligands, and thus inhibited by the ligand blocking activity of amivantamab. The lack of species cross-reactivity with the MET ligand HGF, likely underestimates MET signaling dependencies in these mouse models, although this may play a role in patient tumors. These findings suggest NSCLC patients with elevated wt EGFR expression and lacking driver mutations may achieve therapeutic benefit from amivantamab treatment. Citation Format: Benjamin Henley, Kristen Chevalier, Matthew Smith, Smruthi Vijayaraghaven, Barbara Bushey, Gerald Chu, Joshua Curtin, Nataša Obermajer, Kathryn Packman, Sylvie Laquerre, Matthew Lorenzi, Eric Haura, Sheri Moores. Efficacy of amivantamab, a bispecific EGFR/MET antibody, correlates with EGFR expression and signaling in NSCLC models with wild-type EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 953.

Dissecting strategies to tune the therapeutic potential of SARS-CoV-2-specific monoclonal antibody CR3022.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coupled with a lack of therapeutics, has paralyzed the globe. Although significant effort has been invested in identifying antibodies that block infection, the ability of antibodies to target infected cells through Fc interactions may be vital to eliminate the virus. To explore the role of Fc activity in SARS-CoV-2 immunity, the functional potential of a cross–SARS-reactive antibody, CR3022, was assessed. CR3022 was able to broadly drive antibody effector functions, providing critical immune clearance at entry and upon egress. Using selectively engineered Fc variants, no protection was observed after administration of WT IgG1 in mice or hamsters. Conversely, the functionally enhanced Fc variant resulted in increased pathology in both the mouse and hamster models, causing weight loss in mice and enhanced viral replication and weight loss in the more susceptible hamster model, highlighting the pathological functions of Fc-enhancing mutations. These data point to the critical need for strategic Fc engineering for the treatment of SARS-CoV-2 infection.

Glycine 236 in the Lower Hinge Region of Human IgG1 Differentiates FcγR from Complement Effector Function.

Abs of the IgG isotype mediate effector functions like Ab-dependent cellular cytotoxicity and Ab-dependent cellular phagocytosis by Fc interactions with FcγRs and complement-dependent cytotoxicity upon IgG-Fc binding to C1q. In this study, we describe the crucial role of the highly conserved dual glycines at position 236-237 in the lower hinge region of human IgG, including the lack of one glycine as found in IgG2. We found several permutations in this region that either silence or largely abrogate FcγR binding and downstream FcγR effector functions, as demonstrated by surface plasmon resonance, Ab-dependent cellular phagocytosis, and Ab-dependent cellular cytotoxicity assays. Although the binding regions of FcγRs and C1q on the IgG-Fc largely overlap, IgG1 with a deletion of G236 only silences FcγR-mediated effector functions without affecting C1q-binding or activation. Several mutations resulted in only residual FcγRI binding with differing affinities that are either complement competent or silenced. Interestingly, we also found that IgG2, naturally only binding FcγRIIa, gains binding to FcγRI and FcγRIIIa after insertion of G236, highlighting the crucial importance of G236 in IgG for FcγR interaction. These mutants may become invaluable tools for FcγR-related research as well as for therapeutic purposes in which only complement-mediated functions are required without the involvement of FcγR.

Fc-Engineering for Modulated Effector Functions-Improving Antibodies for Cancer Treatment.

The majority of monoclonal antibody (mAb) therapeutics possess the ability to engage innate immune effectors through interactions mediated by their fragment crystallizable (Fc) domain. By delivering Fc-Fc gamma receptor (FcγR) and Fc-C1q interactions, mAb are able to link exquisite specificity to powerful cellular and complement-mediated effector functions. Fc interactions can also facilitate enhanced target clustering to evoke potent receptor signaling. These observations have driven decades-long research to delineate the properties within the Fc that elicit these various activities, identifying key amino acid residues and elucidating the important role of glycosylation. They have also fostered a growing interest in Fc-engineering whereby this knowledge is exploited to modulate Fc effector function to suit specific mechanisms of action and therapeutic purposes. In this review, we document the insight that has been generated through the study of the Fc domain; revealing the underpinning structure-function relationships and how the Fc has been engineered to produce an increasing number of antibodies that are appearing in the clinic with augmented abilities to treat cancer.