Wild-type IgG1 Research Articles

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...

Abstract 2668: CBO421: A novel drug Fc-conjugate to prevent tumor immune evasion via the CD73/adenosine pathway

Abstract Background CD73 (5’-nucleotidase or NT5E) is a cell surface enzyme responsible for the rate limiting step in adenosine production; the conversion of adenosine monophosphate (AMP) into adenosine. Adenosine contributes to immune evasion by solid tumors by concentrating immunosuppressive adenosine in the tumor microenvironment. CBO421, a Drug Fc-Conjugate (DFC), comprises a novel small molecule CD73 inhibitor stably conjugated to an immune-silent human IgG1 Fc, combining the strengths of small molecule inhibitors and CD73-targeting monoclonal antibodies (mAbs) for potential best-in-class efficacy. Methods CBO421’s binding affinity to human CD73 and Fc receptors was assessed by surface plasmon resonance. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated using a commercial kit. Immunophenotyping and binding to cancer cells was measured by flow cytometry. Inhibition of soluble and membrane-bound CD73 on human PBMCs and mouse cancer cells was determined by measuring free phosphate levels. Functional activity was measured using PBMC rescue assays. In vivo efficacy of CBO421 monotherapy was evaluated in a syngeneic mouse model. Results CBO421 exhibited a binding affinity to human CD73 (KD = 0.8 nM) that was comparable to or greater to the affinity observed with small molecule inhibitors (AB680, OP-5244) and CD73-targeting mAbs (oleclumab, mupadolimab biosimilars). As expected, human FcRn binding of CBO421 was comparable to the unconjugated Fc and the full-length wild-type IgG1 control mAb. CBO421 binding to human Fc gamma receptors and ADCC activity was significantly reduced or abolished compared to the control mAb, such that it poses a minimal risk for undesired autoimmune activity. CBO421 potently inhibited soluble CD73 and CD73 expressed on human MDA-MB-231 and mouse EMT6 cancer cells. Immunophenotyping revealed that CD73 expression was primarily observed on CD8+ T and B cells from human PBMCs. In functional PBMC rescue assays, CBO421 demonstrated potent reactivation of AMP- or ATP-suppressed CD4+ or CD8+ T cells at levels comparable to small molecule inhibitors and greater than anti-CD73 mAb comparators. CBO421 monotherapy demonstrated a dose-dependent reduction in tumor volume in a syngeneic mouse model using MC38, a colorectal cancer cell line. Conclusions CBO421 exhibited strong binding and potent inhibition of both soluble and membrane-bound CD73, differentiating it from mAb CD73 inhibitors. The proprietary immune-silent Fc of CBO421 did not affect FcRn binding but substantially reduced Fc gamma receptor binding, leading to the elimination of ADCC activity to prevent undesired immune cell depletion in host tissues. CBO421 demonstrated high potency in functional cell-based assays and robust antitumor efficacy in a syngeneic mouse model. Currently, CBO421 is being advanced as a clinical development candidate for the treatment of solid cancers. Citation Format: Amanda Almaguer, Doug Zuill, Simon Döhrmann, James Levin, Nicholas Dedeic, Elizabeth Abelovski, Joanne Fortier, Qiping Zhao, Maria Hernandez, Karin Amundson, Madison Moniz, Hongyuan Chen, Dhanya Panickar, Thanh Lam, Tom Brady, Allen Borchardt, Grayson Hough, Jeffrey B. Locke, Jason N. Cole, Leslie W. Tari. CBO421: A novel drug Fc-conjugate to prevent tumor immune evasion via the CD73/adenosine pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2668.

Plant-derived Durvalumab variants show efficient PD-1/PD-L1 blockade and therapeutically favourable FcR binding.

Immune checkpoint blocking therapy targeting the PD-1/PD-L1 inhibitory signalling pathway has produced encouraging results in the treatment of a variety of cancers. Durvalumab (Imfinzi®) targeting PD-L1 is currently used for immunotherapy of several tumour malignancies. The Fc region of this IgG1 antibody has been engineered to reduce FcγR interactions with the aim of enhancing blockade of PD-1/PD-L1 interactions without the depletion of PD-L1-expressing immune cells. Here, we used Nicotiana benthamiana to produce four variants of Durvalumab (DL): wild-type IgG1 and its 'Fc-effector-silent' variant (LALAPG) carrying further modifications to increase antibody half-life (YTE); IgG4S228P and its variant (PVA) with Fc mutations to decrease binding to FcγRI. In addition, DL variants were produced with two distinct glycosylation profiles: afucosylated and decorated with α1,6-core fucose. Plant-derived DL variants were compared to the therapeutic antibody regarding their ability to (i) bind to PD-L1, (ii) block PD-1/PD-L1 inhibitory signalling and (iii) engage with the neonatal Fc receptor (FcRn) and various Fcγ receptors. It was found that plant-derived DL variants bind to recombinant PD-L1 and to PD-L1 expressed in gastrointestinal cancer cells and are able to effectively block its interaction with PD-1 on T cells, thereby enhancing their activation. Furthermore, we show a positive impact of Fc amino acid mutations and core fucosylation on DL's therapeutic potential. Compared to Imfinzi®, DL-IgG1 (LALAPG) and DL-IgG4 (PVA)S228P show lower affinity to CD32B inhibitory receptor which can be therapeutically favourable. Importantly, DL-IgG1 (LALAPG) also shows enhanced binding to FcRn, a key determinant of serum half-life of IgGs.

Multi-Attribute Monitoring Method for Process Development of Engineered Antibody for Site-Specific Conjugation.

Antibody drug conjugates, a class of biotherapeutic proteins, have been extensively developed in recent years, resulting in new approvals and improved standard of care for cancer patients. Among the numerous strategies of conjugating cytotoxic payloads to monoclonal antibodies, insertion of a cysteine residue achieves a tightly controlled, site-specific drug to antibody ratio. Tailored analytical tools are required to direct the development of processes capable of manufacturing novel antibody scaffolds with the desired product quality. Here, we describe the development of a 12 min, mass-spectrometry-based method capable of monitoring four distinct quality attributes simultaneously: variations in the thiol state of the inserted cysteines, N-linked glycosylation, reduction of interchain disulfide bonds, and polypeptide fragmentation. This method provides new insight into the properties of the antibody intermediate and associated manufacturing processes. Oxidized thiol states are formed within the bioreactor, of which a variant containing an additional disulfide bond was produced and remained relatively constant throughout the fed-batch process; reduced thiol variants were introduced upon harvest. Nearly 20 percent of N-linked glycans contained sialic acid, substantially higher than anticipated for wildtype IgG1. Lastly, previously unreported polypeptide fragmentation sites were identified in the C239i constant domain, and the relationship between fragmentation and glycoform were explored. This work illustrates the utility of applying a high-throughput liquid chromatography-mass spectrometry multi-attribute monitoring method to support the development of engineered antibody scaffolds.

A new mouse model expressing human ACE2, TMPRSS2, and FCGRT for evaluating human antibody responses to SARS-CoV-2

Abstract Protection against viruses is most often measured by levels of plasma binding and neutralizing antibodies (Abs) in vitro, which do not capture the full picture of Ab immunity, including Fc functions. The continuing evolution of SARS-CoV-2 underscores the need to understand protection mediated by Abs. We generated triple knock-in (TKI) mice expressing human ACE2, TMPRSS2, and FCGRT in place of murine genes. A human anti-SARS-CoV-2 IgG1 with a half-life extending mutation but not wild-type IgG1 exhibited long half-life and protected against BA.2 infection in lungs, thereby validating our TKI mice for evaluating human Ab clearance and function. We next evaluated protective efficacy of plasma samples from vaccinated San Diegans against Delta or BA.5 by performing passive transfer experiments. Treatment with pooled plasma from vaccination plus infection but not triple vaccination individuals cleared infectious Delta in lungs of TKI mice, although Delta-binding and -neutralizing Ab titers in vitro were similar between the two groups of people. In contrast, neither group’s plasma impacted lung viral burden upon BA.5 challenge. Thus, Abs elicited by vaccination plus infection but not triple vaccination reduce Delta infection in this mouse model, and Abs from both groups are ineffective against BA.5. These results are consistent with epidemiological data, and show that in vitro Ab titers do not predict in vivo protection. Our TKI mouse model represents an important tool to test efficacy of human monoclonal Abs against SARS-CoV-2/related CoVs, predict human polyclonal Ab-mediated protection against newly emerging variants/CoVs, and dissect mechanisms of Ab-mediated protection in vivo. Results will aid development of pan CoV vaccines.

Mechanistic incorporation of FcRn binding in plasma and endosomes in a whole body PBPK model for large molecules.

Monoclonal antibodies, endogenous IgG, and serum albumin bind to FcRn in the endosome for salvaging and recycling after pinocytotic uptake, which prolongs their half-life. This mechanism has been broadly recognized and is incorporated in currently available PBPK models. Newer types of large molecules have been designed and developed, which also bind to FcRn in the plasma space for various mechanistic reasons. To incorporate FcRn binding affinity in PBPK models, binding in the plasma space and subsequent internalisation into the endosome needs to be explicitly represented. This study investigates the large molecules model in PK-Sim® and its applicability to molecules with FcRn binding affinity in plasma. With this purpose, simulations of biologicals with and without plasma binding to FcRn were performed with the large molecule model in PK-Sim®. Subsequently, this model was extended to ensure a more mechanistic description of the internalisation of FcRn and the FcRn-drug complexes. Finally, the newly developed model was used in simulations to explore the sensitivity for FcRn binding in the plasma space, and it was fitted to an in vivo dataset of wild-type IgG and FcRn inhibitor plasma concentrations in Tg32 mice. The extended model demonstrated a strongly increased sensitivity of the terminal half-life towards the plasma FcRn binding affinity and could successfully fit the in vivo dataset in Tg32 mice with meaningful parameter estimates.

Pre-existing Reactivity to an IgG4 Fc-Epitope: Characterization and Mitigation of Interference in a Bridging Anti-drug Antibody Assay

Twenty percent of baseline patient samples exhibited a pre-existing response in a bridging anti-drug antibody (ADA) assay for a human IgG4 monoclonal antibody (mAb) therapeutic. In some cases, assay signals were more than 100-fold higher than background, potentially confounding detection of true treatment-emergent ADA responses. The pre-existing reactivity was mapped by competitive inhibition experiments using recombinant proteins or chimeric human mAbs with IgG4 heavy chain regions swapped for IgG1 sequences. These experiments demonstrated that the majority of the samples had reactivity to an epitope containing leucine 445 in the CH3 domain of human IgG4. The pre-existing reactivity in baseline patient samples was mitigated by replacing the ADA assay capture reagent with a version of the drug containing a wild type IgG1 proline substitution at residue 445 without impacting detection of drug-specific, treatment-emergent ADA. Finally, purification on Protein G or anti-human IgG (H + L) columns indicated the pre-existing response was likely due to immunoglobulins in patient samples.Graphical abstract

Cross-species higher sensitivities of FcγRIIIA/FcγRIV to afucosylated IgG for enhanced ADCC.

BackgroundExpressing afucosylated human IgG1 antibodies with Chinese hamster ovary (CHO) cells deficient of α-(1,6)-fucosyltransferase (FUT8) is being more and more accepted as a routine method to enhance antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies, especially for anti-cancer regimens. However, in pre-clinical studies relying on disease models other than mice and primates, e.g., those underrepresented species for infectious diseases, it is less clear whether such afucosylated antibodies can demonstrate enhanced therapeutic index. This is because the orthologues of human FcγRIIIA or mouse FcγRIV from those species have not been well characterized.MethodsWe set up a luciferase-based ADCC assay with Jurkat reporter cells expressing FcγRIIIA/FcγRIV from human, mouse, rat, hamster, guinea pig, ferret, rabbit, cat, dog, pig and monkey, and also produced human, mouse, hamster, rabbit and pig IgG from wild type and Fut8−/− CHO cells or hybridomas.ResultsWe confirmed that enhanced stimulation through FcγRIIIA/FcγRIV by afucosylated IgG, as compared with wild type IgG, is a cross-species phenomenon.ConclusionsThus, efficacy and toxicology studies of the next generation afucosylated therapeutic IgG and Fc fusion proteins in these underrepresented animal models should be expected to generate translatable data for treating human diseases, leading to the expanded applications of this new class of glycoengineered biologics.

Dreamm-5 Platform Trial: Belantamab Mafodotin (Belamaf) in Combination with Four Different Novel Agents in Patients with Relapsed/Refractory Multiple Myeloma (RRMM)

Dreamm-5 Platform Trial: Belantamab Mafodotin (Belamaf) in Combination with Four Different Novel Agents in Patients with Relapsed/Refractory Multiple Myeloma (RRMM)

IBI112, a selective anti-IL23p19 monoclonal antibody, displays high efficacy in IL-23-induced psoriasiform dermatitis

Psoriasis is a highly prevalent inflammatory skin disease. Plaque psoriasis is the most common type of psoriasis, and the interleukin (IL)-23/IL-17 axis plays a key role in disease progression. In this article, we describe IBI112, a highly potent anti-IL-23 monoclonal antibody under clinical development, which efficiently neutralizes IL23p19, a subunit of IL-23, to abrogate IL-23 binding to its receptor and block downstream signal transducer and activator of transcription 3 (STAT3) phosphorylation. Specifically, IBI112 blocked IL-23 induced downstream IL-17 production from splenocytes. In addition, IBI112 administration reduced skin thickness in a psoriasis-like epidermal hyperplasia mouse model challenged by continuous hIL-23 injection. IBI112 showed synergism with an anti-IL-1R antibody in controlling disease progression in an imiquimod (IMQ) -induced psoriasis model. Moreover, with mutations in Fc fragment of IBI112, extended half-life was observed when compared to the wild-type IgG1 version in both human-FcRn-knock-in mice and cynomolgus monkeys. IBI112 was well tolerated after high dose administration in cynomolgus monkeys. In summary, we have developed an extended half-life, anti-IL-23p19 monoclonal antibody, IBI112, which efficiently neutralized IL-23, blocked IL-23-induced IL-17 production, and alleviated disease symptoms in two mouse models of psoriasis.

Whole-Body Pharmacokinetics of Antibody in Mice Determined using Enzyme-Linked Immunosorbent Assay and Derivation of Tissue Interstitial Concentrations

Here we have reported whole-body disposition of wild-type IgG and FcRn non-binding IgG in mice, determined using Enzyme-Linked Immunosorbent Assay (ELISA). The disposition data generated using ELISA are compared with previously published biodistribution data generated using radiolabelled IgG. In addition, we introduce a novel concept of ABCIS values, which are defined as percentage ratios of tissue interstitial and plasma AUC values. These values can help in predicting tissue interstitial concentrations of monoclonal antibodies (mAbs) based on the plasma concentrations. Tissue interstitial concentrations derived from our study are also compared with previously reported values measured using microdialysis or centrifugation method. Lastly, the new set of biodistribution data generated using ELISA are used to refine the PBPK model for mAbs.

DREAMM-5 platform trial: Belantamab mafodotin in combination with novel agents in patients with relapsed/refractory multiple myeloma (RRMM).

TPS8552 Background: Single-agent belantamab mafodotin (GSK2857916), a B-cell maturation antigen targeting immunoconjugate, induced deep and durable responses in patients with RRMM, with a manageable safety profile (DREAMM-2, NCT03525678, Lancet Oncol.2020). A platform trial design allows efficient evaluation of belantamab mafodotin in combination with other anti-myeloma agents, such as a humanized wild-type IgG1 anti-OX40 agonist, an IgG4 inducible T-cell costimulatory (ICOS) agonist, and a gamma-secretase inhibitor. The unique, multimodal mechanisms of action (MoAs) of belantamab mafodotin, in combination with MoAs of these agents, has the potential to achieve synergistic effects in MM, to further enhance anti-myeloma activity without compromising safety. Methods: DREAMM-5 (NCT04162210) utilizes a master protocol with separate sub-studies comprised of sequential dose exploration (DE) and cohort expansion (CE) phases, to identify promising, effective belantamab mafodotin combinations when compared with a shared belantamab mafodotin monotherapy control arm (CE phase only). DE phases consist of multiple dosing cohorts with belantamab mafodotin combinations where patients are assigned to treatment slots by predetermined algorithmic approach (modified toxicity probability interval; N≤10 per cohort). Recommended phase 2 doses (RP2D) will be based on dose-limiting toxicities, safety, and pharmacokinetics. Interim analyses, based on overall response rate (ORR), determine if a RP2D is moved forward to a CE phase (N≥35 per cohort). Patients in the CE (stratified by number of prior therapies) will be randomized equally to open sub-studies. Eligible patients will have received ≥3 prior therapy lines which must include ≥1 immunomodulatory agent, proteasome inhibitor, and anti-CD38 antibody. The primary objectives are to identify RP2D for each combination (DE phase) and ORR (≥partial response, CE phase). Sub-studies 1 (combination with GSK3174998, OX40 agonist antibody) and 2 (combination with GSK3359609, ICOS agonist antibody) are currently enrolling. Sub-study 3 (combination with nirogacestat [PF-03084014; SpringWorks Therapeutics]) is projected to begin enrollment in the first half of 2020. Additional sub-studies will be explored based on scientific rationale and/or preclinical combination study results. Funding: GlaxoSmithKline (207495). Drug linker technology licensed from Seattle Genetics; monoclonal antibody produced using POTELLIGENT Technology licensed from BioWa. Clinical trial information: NCT04162210 .

Dreamm-5: Platform Trial Evaluating Belantamab Mafodotin (a BCMA-directed Immuno-conjugate) in Combination with Novel Agents in Relapsed or Refractory Multiple Myeloma (RRMM)

Background: Patients with RRMM who have relapsed through multiple prior lines of therapy need novel, effective, targeted agents. B-cell maturation antigen (BCMA) is a cell-surface receptor required for plasma cell survival that is ubiquitously expressed on MM cells, but virtually absent on naïve and memory B cells. Belantamab mafodotin (GSK2857916) is a BCMA-directed immuno-conjugate with an afucosylated, humanized anti-BCMA monoclonal antibody (mAb) conjugated by a protease-resistant cysteine linker to a microtubule disrupting agent, monomethyl auristatin F (MMAF). Belantamab mafodotin specifically binds to BCMA, eliminating MM cells by a multimodal mechanism including delivering MMAF to BCMA-expressing malignant cells, enhancing antibody-dependent cellular cytotoxicity, and leveraging immunogenic cell death. In an open-label, phase 1 study (DREAMM-1), belantamab mafodotin monotherapy had a manageable safety profile and demonstrated rapid, deep and durable clinical response (60% overall response rate, 14.3 months duration of response) with significant progression free-survival (PFS; 12 months) in patients with heavily pretreated MM. Belantamab mafodotin is being evaluated in clinical trials in various lines of MM therapy, either as monotherapy or in combination. The DREAMM-5 platform trial is a phase 1/2 study that incorporates an efficient design with 1 master protocol, wherein multiple belantamab mafodotin-containing combinations will be evaluated in separate substudies to identify effective doublet combinations versus a shared belantamab mafodotin monotherapy control arm. Initial substudies will include combinations with the T-cell activating checkpoint mAbs GSK3359609 (an IgG4 inducible T-cell costimulatory [ICOS] agonist antibody that is Fc optimized to selectively enhance T-cell function to enable antitumor responses), GSK3174998 (a humanized wild-type IgG1 anti-OX40 agonistic mAb), and a gamma-secretase inhibitor, Nirogacestat (PF-03084014, SpringWorks Therapeutics). Furthermore, as treatment paradigms evolve, including potential genomic subsets of patients with RRMM and actionable mutations, other control arms may be introduced. The combination agent for evaluation in each substudy will be selected based on scientific rationale and/or results of preclinical experiments with the selected agent in combination with belantamab mafodotin. Methods: Patients with RRMM will be eligible if they have received ≥3 prior lines of therapy (consisting of an immunomodulatory drug, proteasome inhibitor, and an anti-CD38 mAb), have measurable disease (as measured by M-protein and free light chain), and acceptable hematologic and vital organ functions. Participants previously treated with an anti-BCMA targeted therapy are eligible except for prior belantamab mafodotin treatment and chimeric antigen receptor (CAR) T-cell therapy within 3 months of screening. Each substudy will have 2 sequential phases to evaluate safety and efficacy (Figure). The dose exploration (DE) phase will evaluate the safety and tolerability of belantamab mafodotin administered in combination with a partner agent. Each DE phase will consist of multiple dosing cohorts (N≤10 per cohort), 1 cohort per doublet combination. The primary objective is to identify a recommended phase 2 dose for each doublet combination based on safety and preliminary efficacy. Once a substudy is selected for the cohort expansion (CE) phase (N=35 per substudy), the objective is to compare the response rate between the doublet combination and the shared belantamab mafodotin monotherapy control arm. Secondary objectives in CE are to assess AEs, durability of response, PFS, and overall survival. Exploratory objectives include assessment of PK for belantamab mafodotin with each partner agent, bone marrow minimal residual disease status, and plasma soluble BCMA levels as candidate prognostic and predictive biomarkers. DREAMM-5 is designed to efficiently identify novel, highly effective belantamab mafodotin-containing doublet combinations for evaluation in pivotal trials against current standard-of-care MM agents. Acknowledgments: Editorial assistance provided by Sarah Hauze, PhD, at Fishawack Indicia Ltd and funded by GSK. Study funded by GSK (208887); drug linker technology licensed from Seattle Genetics; monoclonal antibody produced using POTELLIGENT Technology licensed from BioWa. Figure Disclosures Richardson: Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Biswas:GSK: Employment, Equity Ownership. Holkova:GlaxoSmithKline: Employment, Equity Ownership. Jackson:GlaxoSmithKline: Employment, Equity Ownership. Netherway:GlaxoSmithKline: Employment, Equity Ownership. Bao:GSK: Employment, Equity Ownership. Ferron-Brady:GlaxoSmithKline: Employment, Equity Ownership. Yeakey:GlaxoSmithKline: Employment, Equity Ownership. Shelton:GlaxoSmithKline: Employment, Equity Ownership. Montes De Oca:GlaxoSmithKline: Employment. Ahlers:GlaxoSmithKline: Employment, Equity Ownership. Franco:GlaxoSmithKline: Employment, Equity Ownership. Ballas:GlaxoSmithKline: Employment, Equity Ownership; Bristol-Myers Squibb: Equity Ownership; AstraZeneca: Patents & Royalties: Uncompensated copyrights. Paul:GlaxoSmithKline: Employment, Equity Ownership. Luptakova:GlaxoSmithKline: Employment, Equity Ownership. Gupta:GlaxoSmithKline: Employment, Equity Ownership. Opalinska:GSK: Employment, Equity Ownership.

Preliminary safety, efficacy, and pharmacokinetics (PK) results of KN046 (bispecific anti-PD-L1/CTLA4) from a first-in-human study in subjects with advanced solid tumors.

2554 Background: KN046 is a novel bispecific antibody that blocks both PD-L1 interaction with PD1 and CTLA-4 interaction with CD80/CD86. KN046 has a wild type IgG1 Fc portion that preserves intact effector functions, such as depletion of Tregs in tumor microenvironments. This first-in-human study evaluated the safety, tolerability, PK and preliminary efficacy of KN046 in subjects with advanced solid tumors. Methods: This traditional “3+3” dose-escalation design study enrolled patients (pts) with advanced unresectable or metastatic solid tumors refractory or intolerant to standard therapies. Previous treatment from PD1 or PD-L1 immune checkpoint inhibitors was allowed. KN046 was administered intravenously Q2W. Dose limit toxicity (DLT) evaluation period is 28 days. The planned dose levels (DL) were 0.3, 1, 3, 5 and 10 mg/kg. Efficacy evaluation was performed by RECIST 1.1 every 8 weeks. Results: As of Dec 13, 2018, 10 pts had been enrolled (0.3 mg/kg, n = 1; 1 mg/kg, n = 3; 3 mg/kg, n = 3; and 5 mg/kg, n = 3). Median duration of treatment was 8 (range: 2-24) weeks. 1 DLT was observed at 5 mg/kg dose (a grade 3 immune-related hepatitis without elevation in total bilirubin; reversible in two weeks). The most common (≥30%) treatment-emergent AEs (TEAE) were Fatigue, Diarrhea, Nausea, Vomiting. Six immune-related TEAEs (Abdominal pain lower, Arthralgia, Hepatic function abnormal, Hyperthyroidism, Nausea and Transaminitis) were observed in 3 pts. One pt with NSCLC from 3 mg/kg cohort had confirmed completed response. Two pts (TNBC and nivolumab refractory RCC) from 1 mg/kg cohort had shown long-term stable disease ( > 12 weeks). Faster clearance of KN046 was observed at lower dose might be due to target-mediated clearance. T1/2 is approximately 7~9 days at doses of 3 mg/kg and above when saturation occurs. Conclusions: Single agent KN046 has an acceptable safety profile and is in line with previously reported safety data from other immune checkpoint inhibitors. Preliminary efficacy results are promising. PK data from initial 4 cohorts support Q2W schedule. The study is currently ongoing at dose level of 5 mg/kg Q2W. Clinical trial information: NCT03529526.

An Engineered Human Fc variant With Exquisite Selectivity for FcγRIIIaV158 Reveals That Ligation of FcγRIIIa Mediates Potent Antibody Dependent Cellular Phagocytosis With GM-CSF-Differentiated Macrophages.

IgG antibodies mediate the clearance of target cells via the engagement of Fc gamma receptors (FcγRs) on effector cells by eliciting antibody-dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP, respectively). Because (i) the IgG Fc domain binds to multiple FcγRs with varying affinities; (ii) even low Fc:FcγRs affinity interactions can play a significant role when antibodies are engaged in high avidity immune complexes and (iii) most effector cells express multiple FcγRs, the clearance mechanisms that can be mediated by individual FcγR are not well-understood. Human FcγRIIIa (hFcγRIIIa; CD16a), which exists as two polymorphic variants at position 158, hFcγRIIIaV158 and hFcγRIIIaF158, is widely considered to only trigger ADCC, especially with natural killer (NK) cells as effectors. To evaluate the role of hFcγRIIIa ligation in myeloid-derived effector cells, and in particular on macrophages and monocytes which express multiple FcγRs, we engineered an aglycosylated engineered human Fc (hFc) variant, Fc3aV, which binds exclusively to hFcγRIIIaV158. Antibodies formatted with the Fc3aV variant bind to the hFcγRIIIaV158 allotype with a somewhat lower KD than their wild type IgG1 counterparts, but not to any other hFcγR. The exceptional selectivity for hFcγRIIIaV158 was demonstrated by SPR using increased avidity, dimerized GST-fused versions of the ectodomains of hFcγRs and from the absence of binding of large immune complex (IC) to CHO cells expressing each of the hFcγRs, including notably, the FcγRIIIaF158 variant or the highly homologous FcγRIIIb. We show that even though monocyte-derived GM-CSF differentiated macrophages express hFcγRIIIa at substantially lower levels than the other two major activating receptors, namely hFcγRI or hFcγRIIa, Fc3aV-formatted Rituximab and Herceptin perform ADCP toward CD20- and Her2-expressing cancer cells, respectively, at a level comparable to that of the respective wild-type antibodies. We further show that hFcγRIIIa activation plays a significant role on ADCC by human peripheral monocytes. Our data highlight the utility of Fc3aV and other similarly engineered exquisitely selective, aglycosylated Fc variants toward other hFcγRs as tools for the detailed molecular understanding of hFcγR biology.

Targeting CD45 with an Amanitin Antibody-Drug Conjugate Effectively Depletes Human HSCs and Immune Cells for Transplant Conditioning

IntroductionBone Marrow Transplant (BMT) is a potentially curative treatment for malignant and non-malignant blood disorders and has demonstrated impressive outcomes in autoimmune diseases. Prior to BMT, patients are prepared with high-dose chemotherapy alone or with total body irradiation, and both are associated with early and late morbidities, such as infertility, secondary malignancies and organ toxicity; and substantial risk of mortality. This greatly limits the use of BMT in malignant and non-malignant conditions. To address these issues, we are developing antibody drug conjugates (ADCs) targeting hematopoietic stem cells (HSCs) and immune cells to more safely condition patients for BMT.ResultsTo enable simultaneous HSC and immune cell depletion for BMT we investigated targeting human CD45, a protein expressed exclusively on nearly all blood cells including HSCs. Antibody discovery campaigns identified several antibodies with sub-nanomolar affinities for human and non-human primate (NHP) CD45. We then created anti-CD45 ADCs with drug payloads including DNA-damaging, tubulin-targeting and RNA polymerase-inhibiting molecules. An ADC developed with alpha-amanitin (an RNA polymerase II inhibitor) enabled potent in vitro killing of primary human CD34+ HSCs and immune cells (40-120 picomolar IC50s).With this anti-CD45 amanitin ADC (CD45-AM), we explored depletion of HSCs and immune cells in vivo using humanized NSG mice. A single dose of 1 or 3 mg/kg CD45-AM enabled >95% depletion of human CD34+ cells in the bone marrow as assessed 7 or 14 days post-administration (Figure, n = 3/group, p values < 0.05); >95% depletion of human B-, T- and myeloid cells was observed in the periphery and bone marrow (Figure, p values < 0.05). Control non-targeting isotype matched-ADCs and anti-CD45 antibody not bearing a toxin had minimal effect on either HSC or immune cells.In hematopoietic malignancies, an anti-CD45 ADC would ideally reduce disease burden and enable BMT. In a model of acute lymphoblastic leukemia (REH cell line, n = 10 mice/group), and 3 patient-derived models of FLT3+NPM1+ acute myeloid leukemia (n = 4-5 mice/group per model), a single dose of 1 mg/kg CD45-AM more than doubled the median survival and several mice survived disease-free (p values < 0.001).Anti-CD45 antibodies have been investigated for BMT conditioning in patients as naked antibodies that rely on Fc-effector function to deplete lymphocytes (Biol Blood Marrow Transplant. 2003 9(4): 273-81); or as radioimmunotherapy (Blood. 2006 107(5): 2184-2191). These agents demonstrated infusion-related toxicities likely due to effector function elicited by the wild-type IgG backbone. To address this issue, we created anti-CD45 antibodies with reduced Fc-gamma receptor binding that prevented cytokine release in vitro and in humanized mice. As BMT will likely require fast clearing ADCs to avoid depleting the incoming graft, we also created fast-half-life CD45-AM variants with a t½ of 8-15 hours in mice. To determine the safety and pharmacokinetic properties of regular and fast half-life Fc-silent variants in an immune-competent large animal we tested these in cynomolgus monkeys. Single doses (3 mg/kg, iv, n = 3/group) of fast and regular half-life Fc-silent unconjugated anti-CD45 antibodies were both well tolerated in cynomolgus monkeys and displayed pharmacokinetic properties suitable for BMT.ConclusionThese results demonstrate that targeting CD45 with an amanitin ADC results in potent in vitro and in vivo human HSC and immune cell depletion. This new CD45-AM ADC also significantly reduced disease burden in multiple leukemia models. Our results indicate Fc-silencing may avoid infusion-related toxicities observed with previous CD45 mAbs. An alpha-amanitin ADC targeted to CD45 may be appropriate for preparing patients for BMT since we hypothesize it may i) be non-genotoxic; ii) effectively deplete both HSC and immune cells; iii) avoid bystander toxicity, due to amanitin's poor cell permeability as a free toxin; and iv) kill cycling and non-cycling cells, the latter being necessary for effective HSC depletion. As our anti-CD45 ADCs are cross-reactive, we are currently investigating their HSC and immune cell depletion activity in vivo in NHPs to enable further preclinical development of these transplant conditioning agents. [Display omitted] DisclosuresPalchaudhuri:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties; Harvard University: Patents & Royalties. Pearse:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Aslanian:Magenta Therapeutics: Employment, Equity Ownership. McDonough:Magenta Therapeutics: Employment, Equity Ownership. Sarma:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Bhat:Magenta Therapeutics: Employment. Ladwig:Magenta Therapeutics: Employment, Equity Ownership. McShea:Magenta Therapeutics: Employment, Equity Ownership. Kallen:Magenta Therapeutics: Employment, Equity Ownership. Li:Magenta Therapeutics: Employment, Equity Ownership. Panwar:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Dushime:Magenta Therapeutics: Employment, Equity Ownership. Sawant:Magenta Therapeutics: Employment, Equity Ownership. Adams:Magenta Therapeutics: Employment, Equity Ownership. Falahee:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Lamothe:Magenta Therapeutics: Employment, Equity Ownership. Gabros:Magenta Therapeutics: Employment, Equity Ownership. Kien:Magenta Therapeutics: Employment, Equity Ownership. Gillard:Magenta Therapeutics: Employment, Equity Ownership. McDonagh:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Abstract 5761: Identification of Fc mutations that optimize agonistic activity of human anti-CD40 monoclonal antibodies by enhancing appropriate Fcg receptor interactions

Abstract Several agonistic antibodies against human CD40 are currently being explored as immune therapy for cancer patients. Previously, anti-CD40 antibody therapy showed some clinical efficacy, but significant toxicity at the efficacious dose limited further development. To develop an anti-CD40 agonistic antibody with an improved clinical therapeutic index, we focused on modifications to the heavy chain isotype that would impact interactions with specific Fc receptors. In mouse models, anti-CD40 murine IgG1 demonstrated stronger immune stimulatory activities than murine IgG2a. The CD40 murine IgG2a antibody induced acute cytokine release within hours of dosing and led to mortality weeks later. To generate human antibodies with an Fc structure that functions similarly to murine IgG1, human IgG1 Fc mutations with reduced binding on human CD16, enhanced binding on CD32A/B, and maintained binding on CD64 were identified through a proprietary mammalian cell surface display technology. A panel of anti-human CD40 antibodies carrying different Fc regions were created. In a CD40 transfected HEK293 NFκB reporter cell assay, where agonistic activity was not related to Fcγ receptor interactions, anti-CD40 human IgG2 demonstrated stronger agonistic activity than human IgG1 antibodies with either wild type or mutated Fc. This result confirmed the reported role of human IgG2 hinge in impacting the agonistic activity of anti-CD40. In assays using primary B cells or monocyte-derived dendritic cells, which constitutively express Fcγ receptors, a subset of anti-CD40 human IgG1 antibodies showed stronger potency than the corresponding human IgG2, due to the better Fcγ receptor crosslinking for IgG1 antibodies. Swapping the human IgG2 hinge into IgG1 Fc did not further enhance the agonistic activity of anti-CD40 antibodies in the primary cell assays, suggesting that the Fc-crosslinking plays a dominant role in mediating the agonistic activity of these anti-CD40 antibodies in biological systems with cells expressing Fcγ receptor. Engineering the proprietary human IgG1 Fc mutations to these anti-CD40 antibodies further enhanced agonistic activity compared to wild type IgG1 in primary cell assays. In addition to the potent agonistic activity, anti-CD40 with the IgG1 Fc mutations demonstrated lower ADCC and reduced cytokine production driven by Fc receptor activation in a co-culture system. Finally, an Fc modified anti-CD40 agonistic antibody demonstrated an acceptable safety profile in cynomolgous monkey toxicity studies. These data suggest anti-CD40 with the proprietary IgG1 Fc mutations represents a newer generation of CD40 agonists for oncology immune therapy. Citation Format: Shiming Ye, Diane Cohen, Donghee Choi, Siu sze Tan, Joanna Xiong, Yoshiko Akamatsu, Debra Chao, Diane Hollenbaugh, Fiona Harding. Identification of Fc mutations that optimize agonistic activity of human anti-CD40 monoclonal antibodies by enhancing appropriate Fcg receptor interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5761.

A Minimal Physiologically Based Pharmacokinetic Model with a Nested Endosome Compartment for Novel Engineered Antibodies.

We proposed here a minimal physiologically based pharmacokinetic (mPBPK) model for a group of novel engineered antibodies in mice and humans. These antibodies are designed with altered binding properties of their Fc domain with neonatal Fc receptor (FcRn) or the Fab domain with their cognate targets (recycling antibodies) in acidic endosomes. To enable simulations of such binding features in the change of antibody pharmacokinetics and its target suppression, we nested an endothelial endosome compartment in parallel with plasma compartment based on our previously established mPBPK model. The fluid-phase pinocytosis rate from plasma to endothelial endosomes was reflected by the clearance of antibodies in FcRn dysfunctional humans or FcRn-knockout mice. The endosomal recycling rate of FcRn-bound antibodies was calculated based on the reported endosomal transit time. The nonspecific catabolism in endosomes was fitted using pharmacokinetic data of a human wild-type IgG1 adalimumab in humans and B21M in human FcRn (hFcRn) transgenic mice. The developed model adequately predicted the pharmacokinetics of infliximab, motavizumab, and an Fc variant of motavizumab in humans and the pharmacokinetics of bevacizumab, an Fc variant of bevacizumab, and a recycling antibody PH-IgG1 and its non-pH dependent counterpart NPH-IgG1 in hFcRn transgenic mice. Our proposed model provides a platform for evaluation of the pharmacokinetics and disposition behaviors of Fc-engineered antibodies and recycling antibodies.

Rapid Purification of Human Bispecific Antibodies via Selective Modulation of Protein A Binding

Methods to rapidly generate high quality bispecific antibodies (BsAb) having normal half-lives are critical for therapeutic programs. Here, we identify 3 mutations (T307P, L309Q, and Q311R or “TLQ”) in the Fc region of human IgG1 which disrupt interaction with protein A while enhancing interaction with FcRn. The mutations are shown to incrementally alter the pH at which a mAb elutes from protein A affinity resin. A BsAb comprised of a TLQ mutant and a wild-type IgG1 can be efficiently separated from contaminating parental mAbs by differential protein A elution starting from either a) purified parental mAbs, b) in-supernatant crossed parental mAbs, or c) co-transfected mAbs. We show that the Q311R mutation confers enhanced FcRn interaction in vitro, and Abs harboring either the Q311R or TLQ mutations have serum half-lives as long as wild-type human IgG1. The mutant Abs have normal thermal stability and Fcγ receptor interactions. Together, the results lead to a method for high-throughput generation of BsAbs suitable for in vivo studies.

A new approach for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1

Bispecific antibodies combine two different antigen-binding sites in a single molecule, enabling more specific targeting, novel mechanisms of action, and higher clinical efficacies. Although they have the potential to outperform conventional monoclonal antibodies, many bispecific antibodies have issues regarding production, stability, and pharmacokinetic properties. Here, we describe a new approach for generating bispecific antibodies using a common light chain format and exploiting the stable architecture of human immunoglobulin G1. We used iterative experimental validation and computational modeling to identify multiple Fc variant pairs that drive efficient heterodimerization of the antibody heavy chains. Accelerated stability studies enabled selection of one Fc variant pair dubbed “DEKK” consisting of substitutions L351D and L368E in one heavy chain combined with L351K and T366K in the other. Solving the crystal structure of the DEKK Fc region at a resolution of 2.3 Å enabled detailed analysis of the interactions inducing CH3 interface heterodimerization. Local shifts in the IgG backbone accommodate the introduction of lysine side chains that form stabilizing salt-bridge interactions with substituted and native residues in the opposite chain. Overall, the CH3 domain adapted to these shifts at the interface, yielding a stable Fc conformation very similar to that in wild-type IgG. Using the DEKK format, we generated the bispecific antibody MCLA-128, targeting human EGF receptors 2 and 3. MCLA-128 could be readily produced and purified at industrial scale with a standard mammalian cell culture platform and a routine purification protocol. Long-term accelerated stability assays confirmed that MCLA-128 is highly stable and has excellent biophysical characteristics.