Differential effects of FcRn antagonists on the subcellular trafficking of FcRn and albumin.

The homeostasis of IgG is maintained by the neonatal Fc receptor, FcRn. Consequently, antagonism of FcRn to reduce endogenous IgG levels is an emerging strategy for treating antibody-mediated autoimmune disorders using either FcRn-specific antibodies or an engineered Fc fragment. For certain FcRn-specific antibodies, this approach has resulted in reductions in the levels of serum albumin, the other major ligand transported by FcRn. Cellular and molecular analyses of a panel of FcRn antagonists have been carried out to elucidate the mechanisms leading to their differential effects on albumin homeostasis. These analyses have identified 2 processes underlying decreases in albumin levels during FcRn blockade: increased degradation of FcRn and competition between antagonist and albumin for FcRn binding. These findings have potential implications for the design of drugs to modulate FcRn function.

Site-specific 68Ga-labeled nanobody for PET imaging of CD70 expression in preclinical tumor models.

BACKGROUND: CD70-CD27 is a costimulatory ligand-receptor pair in the tumor necrosis factor receptor family. With only limited expression in normal tissues, CD70 is constitutively expressed in a variety of solid tumors and hematologic malignancies, facilitating immunosuppression through CD27 signaling in the tumor microenvironment by enhanced survival of regulatory T cells, induction of T cell apoptosis, and T cell exhaustion. Consequently, CD70 is an increasingly recognized target for developing antibody-based therapies, but its expression patterns vary among different tumor types in spatial distribution, magnitude of expression and percentage of positive cells. In that regard, individual confirmation of CD70 expression at screening and during treatment could enhance the successful implementation of anti-CD70 therapies. Here, we developed a gallium-68 (68Ga) radiolabeled single-domain antibody-fragment targeting CD70 for in vivo positron emission tomography (PET) imaging. RESULTS: An anti-CD70 VHH construct containing a C-direct-tag with a free thiol was developed to enable site-specific conjugation to a NOTA bifunctional chelator for 68Ga radiolabeling. [68Ga]Ga-NOTA-anti-CD70 VHH was obtained in good radiochemical yield of 30.4 ± 1.7% and high radiochemical purity (> 94%). The radiolabeled VHH showed excellent in vitro and in vivo stability. Specific binding of [68Ga]Ga-NOTA-anti-CD70 VHH was observed on CD70high 786-O cells, showing significantly higher cell-associated activity when compared to the blocking condition (p < 0.0001) and CD70low NCl-H1975 cells (p < 0.0001). PET imaging showed specific radiotracer accumulation in CD70 expressing human tumor xenografts, which was efficiently blocked by prior injection of unlabeled anti-CD70 VHH (p = 0.0029). In addition, radiotracer uptake in CD70high tumors was significantly higher when compared with CD70low tumors (p < 0.0001). The distribution of the radioactivity in the tumors using autoradiography was spatially matched with immunohistochemistry analysis of CD70 expression. CONCLUSION: [68Ga]Ga-NOTA-anti-CD70 VHH showed excellent in vivo targeting of CD70 in human cancer xenografts. PET imaging using this radioimmunoconjugate holds promise as a non-invasive method to identify and longitudinally follow-up patients who will benefit most from anti-CD70 therapies.

Chemokine switch regulated by TGF-ß1 in cancer-associated fibroblast subsets determines the efficacy of chemo-immunotherapy.

Combining immunogenic cell death-inducing chemotherapies and PD-1 blockade can generate remarkable tumor responses. It is now well established that TGF-ß1 signaling is a major component of treatment resistance and contributes to the cancer-related immunosuppressive microenvironment. However, whether TGF-ß1 remains an obstacle to immune checkpoint inhibitor efficacy when immunotherapy is combined with chemotherapy is still to be determined. Several syngeneic murine models were used to investigate the role of TGF-ß1 neutralization on the combinations of immunogenic chemotherapy (FOLFOX: 5-fluorouracil and oxaliplatin) and anti-PD-1. Cancer-associated fibroblasts (CAF) and immune cells were isolated from CT26 and PancOH7 tumor-bearing mice treated with FOLFOX, anti-PD-1 ± anti-TGF-ß1 for bulk and single cell RNA sequencing and characterization. We showed that TGF-ß1 neutralization promotes the therapeutic efficacy of FOLFOX and anti-PD-1 combination and induces the recruitment of antigen-specific CD8+ T cells into the tumor. TGF-ß1 neutralization is required in addition to chemo-immunotherapy to promote inflammatory CAF infiltration, a chemokine production switch in CAF leading to decreased CXCL14 and increased CXCL9/10 production and subsequent antigen-specific T cell recruitment. The immune-suppressive effect of TGF-ß1 involves an epigenetic mechanism with chromatin remodeling of CXCL9 and CXCL10 promoters within CAF DNA in a G9a and EZH2-dependent fashion. Our results strengthen the role of TGF-ß1 in the organization of a tumor microenvironment enriched in myofibroblasts where chromatin remodeling prevents CXCL9/10 production and limits the efficacy of chemo-immunotherapy.

The Fab region of IgG impairs the internalization pathway of FcRn upon Fc engagement.

Binding to the neonatal Fc receptor (FcRn) extends serum half-life of IgG, and antagonizing this interaction is a promising therapeutic approach in IgG-mediated autoimmune diseases. Fc-MST-HN, designed for enhanced FcRn binding capacity, has not been evaluated in the context of a full-length antibody, and the structural properties of the attached Fab regions might affect the FcRn-mediated intracellular trafficking pathway. Here we present a comprehensive comparative analysis of the IgG salvage pathway between two full-size IgG1 variants, containing wild type and MST-HN Fc fragments, and their Fc-only counterparts. We find no evidence of Fab-regions affecting FcRn binding in cell-free assays, however, cellular assays show impaired binding of full-size IgG to FcRn, which translates into improved intracellular FcRn occupancy and intracellular accumulation of Fc-MST-HN compared to full size IgG1-MST-HN. The crystal structure of Fc-MST-HN in complex with FcRn provides a plausible explanation why the Fab disrupts the interaction only in the context of membrane-associated FcRn. Importantly, we find that Fc-MST-HN outperforms full-size IgG1-MST-HN in reducing IgG levels in cynomolgus monkeys. Collectively, our findings identify the cellular membrane context as a critical factor in FcRn biology and therapeutic targeting.

Effect of FcRn antagonism on protective antibodies and to vaccines in IgG-mediated autoimmune diseases pemphigus and generalised myasthenia gravis.

Antagonism of the neonatal Fc receptor (FcRn) by efgartigimod has been studied in several autoimmune diseases mediated by immunoglobulin G (IgG) as a therapeutic approach to remove pathogenic IgGs. Whereas reduction of pathogenic titres has demonstrated efficacy in multiple autoimmune diseases, reducing total IgG could potentially increase infection risk in patients receiving FcRn antagonists. The objective of this study was to analyse the effect of FcRn antagonism with efgartigimod on existing protective antibody titres and the ability to mount an immune response after vaccine challenge. Serum levels of total IgG and protective antibodies against tetanus toxoid (TT), varicella zoster virus (VZV), and pneumococcal capsular polysaccharide (PCP) were measured in all patients enrolled in an open-label trial of efgartigimod for the treatment of pemphigus. Vaccine specific-responses were assessed by measuring changes in IgG titres in patients with generalised myasthenia gravis (gMG) who were treated with efgartigimod and who received influenza, pneumococcal, or coronavirus disease 2019 (COVID-19) vaccines during participation in the double-blind trial ADAPT or open-label extension, ADAPT+ (n = 17). FcRn antagonism reduced levels of protective anti-TT, anti-VZV, and anti-PCP antibodies and total IgG to a similar extent; anti-TT and anti-VZV titres remained above minimally protective thresholds for the majority of patients, (10/12) 83% and (14/15) 93% respectively. Protective antibodies returned to baseline values upon treatment cessation. Antigen-specific IgG responses to influenza, pneumococcal, and COVID-19 immunisation were detected in patients with gMG who received these vaccines while undergoing therapy with efgartigimod. In conclusion, FcRn antagonism with efgartigimod did not hamper generation of IgG responses but did transiently reduce IgG titres of all specificities.

Directing HIV-1 for degradation by non-target cells, using bi-specific single-chain llama antibodies.

While vaccination against HIV-1 has been so far unsuccessful, recently broadly neutralizing antibodies (bNAbs) against HIV-1 envelope glycoprotein were shown to induce long-term suppression in the absence of antiretroviral therapy in patients with antibody-sensitive viral reservoirs. The requirement of neutralizing antibodies indicates that the antibody mediated removal (clearance) of HIV-1 in itself is not efficient enough in these immune compromised patients. Here we present a novel, alternative approach that is independent of a functional immune system to clear HIV-1, by capturing the virus and redirecting it to non-target cells where it is internalized and degraded. We use bispecific antibodies with domains derived from small single chain Llama antibodies (VHHs). These bind with one domain to HIV-1 envelope proteins and with the other domain direct the virus to cells expressing epidermal growth factor receptor (EGFR), a receptor that is ubiquitously expressed in the body. We show that HIV envelope proteins, virus-like particles and HIV-1 viruses (representing HIV-1 subtypes A, B and C) are efficiently recruited to EGFR, internalized and degraded in the lysosomal pathway at low nM concentrations of bispecific VHHs. This directed degradation in non-target cells may provide a clearance platform for the removal of viruses and other unwanted agents from the circulation, including toxins, and may thus provide a novel method for curing.

The CD70-CD27 axis in oncology: the new kids on the block.

The immune checkpoint molecule CD70 and its receptor CD27 are aberrantly expressed in many hematological and solid malignancies. Dysregulation of the CD70-CD27 axis within the tumor and its microenvironment is associated with tumor progression and immunosuppression. This is in contrast to physiological conditions, where tightly controlled expression of CD70 and CD27 plays a role in co-stimulation in immune responses. In hematological malignancies, cancer cells co-express CD70 and CD27 promoting stemness, proliferation and survival of malignancy. In solid tumors, only expression of CD70 is present on the tumor cells which can facilitate immune evasion through CD27 expression in the tumor microenvironment. The discovery of these tumor promoting and immunosuppressive effects of the CD70-CD27 axis has unfolded a novel target in the field of oncology, CD70.In this review, we thoroughly discuss current insights into expression patterns and the role of the CD70-CD27 axis in hematological and solid malignancies, its effect on the tumor microenvironment and (pre)clinical therapeutic strategies.

Anti-C2 Antibody ARGX-117 Inhibits Complement in a Disease Model for Multifocal Motor Neuropathy.

BACKGROUND AND OBJECTIVES: To determine the role of complement in the disease pathology of multifocal motor neuropathy (MMN), we investigated complement activation, and inhibition, on binding of MMN patient-derived immunoglobulin M (IgM) antibodies in an induced pluripotent stem cell (iPSC)-derived motor neuron (MN) model for MMN. METHODS: iPSC-derived MNs were characterized for the expression of complement receptors and membrane-bound regulators, for the binding of circulating IgM anti-GM1 from patients with MMN, and for subsequent fixation of C4 and C3 on incubation with fresh serum. The potency of ARGX-117, a novel inhibitory monoclonal antibody targeting C2, to inhibit fixation of complement was assessed. RESULTS: iPSC-derived MNs moderately express the complement regulatory proteins CD46 and CD55 and strongly expressed CD59. Furthermore, MNs express C3aR, C5aR, and complement receptor 1. IgM anti-GM1 antibodies in serum from patients with MMN bind to MNs and induce C3 and C4 fixation on incubation with fresh serum. ARGX-117 inhibits complement activation downstream of C4 induced by patient-derived anti-GM1 antibodies bound to MNs. DISCUSSION: Binding of IgM antibodies from patients with MMN to iPSC-derived MNs induces complement activation. By expressing complement regulatory proteins, particularly CD59, MNs are protected against complement-mediated lysis. Yet, because of expressing C3aR, the function of these cells may be affected by complement activation upstream of membrane attack complex formation. ARGX-117 inhibits complement activation upstream of C3 in this disease model for MMN and therefore represents an intervention strategy to prevent harmful effects of complement in MMN.

Cusatuzumab for treatment of CD70-positive relapsed or refractory cutaneous T-cell lymphoma.

BACKGROUND: The clinical benefit of cusatuzumab, a CD70-directed monoclonal antibody with enhanced effector functions, was investigated in patients with relapsed/refractory (R/R) cutaneous T-cell lymphoma (CTCL). METHODS: In this cohort expansion of the ARGX-110-1201 study, 27 patients with R/R CTCL received cusatuzumab at 1 (n = 11) or 5 mg/kg (n = 16) once every 3 weeks to investigate its safety, dose, and exploratory efficacy. The pharmacokinetics, immunogenicity, CD70 expression, and CD70/CD27 biology were also assessed. RESULTS: The most common adverse events included infusion-related reactions, pyrexia, and asthenia. Eighteen serious adverse events (grade 1-3) were reported in 11 patients; 1 of these (vasculitis) was considered drug-related. For 8 of the 11 patients receiving 1 mg/kg, anti-drug antibodies (ADAs) affected the minimal concentration, and this resulted in undetectable cusatuzumab concentrations at the end of treatment and, in some cases, a loss of response. This effect was greatly reduced in the patients receiving 5 mg/kg. The overall response rate was 23%; this included 1 complete response and 5 partial responses (PRs) in 26 of the 27 evaluable patients. In addition, 9 patients achieved stable disease. The mean duration on cusatuzumab was 5.2 months, and the median duration was 2.5 months. Patients with Sézary syndrome (SS) achieved a 60% PR rate with a dosage of 5 mg/kg and a 33% PR rate with a dosage of 1 mg/kg; this resulted in an overall response rate of 50% for patients with SS at both doses. CONCLUSIONS: Cusatuzumab was well tolerated, and antitumor activity was observed at both 1 and 5 mg/kg in highly pretreated patients with R/R CTCL. The observed dose-dependent effect on exposure supports the use of 5 mg/kg for future development.

IgG regulation through FcRn blocking: A novel mechanism for the treatment of myasthenia gravis.

The neonatal Fc receptor (FcRn) is an MHC class I-like molecule that is widely distributed in mammalian organs, tissues, and cells. FcRn is critical to maintaining immunoglobulin G (IgG) and albumin levels through rescuing these molecules from lysosomal degradation. IgG autoantibodies are associated with many autoimmune diseases, including myasthenia gravis (MG), a rare neuromuscular autoimmune disease that causes debilitating and, in its generalized form (gMG), potentially life-threatening muscle weakness. IgG autoantibodies are directly pathogenic in MG and target neuromuscular junction proteins, causing neuromuscular transmission failure. Treatment approaches that reduce autoantibody levels, such as therapeutic plasma exchange and intravenous immunoglobulin, have been shown to be effective for gMG patients but are not indicated as ongoing maintenance therapies and can be associated with burdensome side effects. Agents that block FcRn-mediated recycling of IgG represent a rational and promising approach for the treatment of gMG. Blocking FcRn allows targeted reduction of all IgG subtypes without decreasing concentrations of other Ig isotypes; therefore, FcRn blocking could be a safe and effective treatment strategy for a broad population of gMG patients. Several FcRn-blocking antibodies and one antibody Fc fragment have been developed and are currently in various stages of clinical development. This article describes the mechanism of FcRn blockade as a novel approach for IgG-mediated disease therapy and reviews promising clinical data using such FcRn blockers for the treatment of gMG.

The NHance® Mutation-Equipped Anti-MET Antibody ARGX-111 Displays Increased Tissue Penetration and Anti-Tumor Activity in Advanced Cancer Patients.

Dysregulation of MET signaling has been implicated in tumorigenesis and metastasis. ARGX-111 combines complete blockade of this pathway with enhanced tumor cell killing and was investigated in 24 patients with MET-positive advanced cancers in a phase 1b study at four dose levels (0.3-10 mg/kg). ARGX-111 was well tolerated up to 3 mg/kg (MTD). Anti-tumor activity was observed in nearly half of the patients (46%) with a mean duration of treatment of 12 weeks. NHance® mutations in the Fc of ARGX-111 increased affinity for the neonatal Fc receptor (FcRn) at acidic pH, stimulating transcytosis across FcRn-expressing cells and radiolabeled ARGX-111 accumulated in lymphoid tissues, bone and liver, organs expressing FcRn at high levels in a biodistribution study using human FcRn transgenic mice. In line with this, we observed, in a patient with MET-amplified (>10 copies) gastric cancer, diminished metabolic activity in multiple metastatic lesions in lymphoid and bone tissues by 18F-FDG-PET/CT after two infusions with 0.3 mg/kg ARGX-111. When escalated to 1 mg/kg, a partial response was reached. Furthermore, decreased numbers of CTC (75%) possibly by the enhanced tumor cell killing witnessed the modes of action of the drug, warranting further clinical investigation of ARGX-111.

VEGFR-1/Flt-1 inhibition increases angiogenesis and improves muscle function in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy is characterized by structural degeneration of muscle, which is exacerbated by localized functional ischemia due to loss of nitric oxide synthase-induced vasodilation. Treatment strategies aimed at increasing vascular perfusion have been proposed. Toward this end, we have developed monoclonal antibodies (mAbs) that bind to the vascular endothelial growth factor (VEGF) receptor VEGFR-1 (Flt-1) and its soluble splice variant isoform (sFlt-1) leading to increased levels of free VEGF and proangiogenic signaling. The lead chimeric mAb, 21B3, had high affinity and specificity for both human and mouse sFlt-1 and inhibited VEGF binding to sFlt-1 in a competitive manner. Proof-of-concept studies in the mdx mouse model of Duchenne muscular dystrophy showed that intravenous administration of 21B3 led to elevated VEGF levels, increased vascularization and blood flow to muscles, and decreased fibrosis after 6-12 weeks of treatment. Greater muscle strength was also observed after 4 weeks of treatment. A humanized form of the mAb, 27H6, was engineered and demonstrated a comparable pharmacologic effect. Overall, administration of anti-Flt-1 mAbs in mdx mice inhibited the VEGF:Flt-1 interaction, promoted angiogenesis, and improved muscle function. These studies suggest a potential therapeutic benefit of Flt-1 inhibition for patients with Duchenne muscular dystrophy.

ARGX-117, a therapeutic complement inhibiting antibody targeting C2.

BACKGROUND: Activation of the classical and lectin pathway of complement may contribute to tissue damage and organ dysfunction of antibody-mediated diseases and ischemia-reperfusion conditions. Complement factors are being considered as targets for therapeutic intervention. OBJECTIVE: We sought to characterize ARGX-117, a humanized inhibitory monoclonal antibody against complement C2. METHODS: The mode-of-action and binding characteristics of ARGX-117 were investigated in detail. Furthermore, its efficacy was analyzed in in vitro complement cytotoxicity assays. Finally, a pharmacokinetic/pharmacodynamic study was conducted in cynomolgus monkeys. RESULTS: Through binding to the Sushi-2 domain of C2, ARGX-117 prevents the formation of the C3 proconvertase and inhibits classical and lectin pathway activation upstream of C3 activation. As ARGX-117 does not inhibit the alternative pathway, it is expected not to affect the antimicrobial activity of this complement pathway. ARGX-117 prevents complement-mediated cytotoxicity in in vitro models for autoimmune hemolytic anemia and antibody-mediated rejection of organ transplants. ARGX-117 exhibits pH- and calcium-dependent target binding and is Fc-engineered to increase affinity at acidic pH to the neonatal Fc receptor, and to reduce effector functions. In cynomolgus monkeys, ARGX-117 dose-dependently reduces free C2 levels and classical pathway activity. A 2-dose regimen of 80 and 20 mg/kg separated by a week, resulted in profound reduction of classical pathway activity lasting for at least 7 weeks. CONCLUSIONS: ARGX-117 is a promising new complement inhibitor that is uniquely positioned to target both the classical and lectin pathways while leaving the alternative pathway intact.

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.

Selective inhibition of TGF-ß1 produced by GARP-expressing Tregs overcomes resistance to PD-1/PD-L1 blockade in cancer.

TGF-ß1, ß2 and ß3 bind a common receptor to exert vastly diverse effects in cancer, supporting either tumor progression by favoring metastases and inhibiting anti-tumor immunity, or tumor suppression by inhibiting malignant cell proliferation. Global TGF-ß inhibition thus bears the risk of undesired tumor-promoting effects. We show that selective blockade of TGF-ß1 production by Tregs with antibodies against GARP:TGF-ß1 complexes induces regressions of mouse tumors otherwise resistant to anti-PD-1 immunotherapy. Effects of combined GARP:TGF-ß1/PD-1 blockade are immune-mediated, do not require FcγR-dependent functions and increase effector functions of anti-tumor CD8+ T cells without augmenting immune cell infiltration or depleting Tregs within tumors. We find GARP-expressing Tregs and evidence that they produce TGF-ß1 in one third of human melanoma metastases. Our results suggest that anti-GARP:TGF-ß1 mAbs, by selectively blocking a single TGF-ß isoform emanating from a restricted cellular source exerting tumor-promoting activity, may overcome resistance to PD-1/PD-L1 blockade in patients with cancer.

Targeting CD70 with cusatuzumab eliminates acute myeloid leukemia stem cells in patients treated with hypomethylating agents.

Acute myeloid leukemia (AML) is driven by leukemia stem cells (LSCs) that resist conventional chemotherapy and are the major cause of relapse1,2. Hypomethylating agents (HMAs) are the standard of care in the treatment of older or unfit patients with AML, but responses are modest and not durable3-5. Here we demonstrate that LSCs upregulate the tumor necrosis factor family ligand CD70 in response to HMA treatment resulting in increased CD70/CD27 signaling. Blocking CD70/CD27 signaling and targeting CD70-expressing LSCs with cusatuzumab, a human αCD70 monoclonal antibody with enhanced antibody-dependent cellular cytotoxicity activity, eliminated LSCs in vitro and in xenotransplantation experiments. Based on these preclinical results, we performed a phase 1/2 trial in previously untreated older patients with AML with a single dose of cusatuzumab monotherapy followed by a combination therapy with the HMA azacitidine ( NCT03030612 ). We report results from the phase 1 dose escalation part of the clinical trial. Hematological responses in the 12 patients enrolled included 8 complete remission, 2 complete remission with incomplete blood count recovery and 2 partial remission with 4 patients achieving minimal residual disease negativity by flow cytometry at <10-3. Median time to response was 3.3 months. Median progression-free survival was not reached yet at the time of the data cutoff. No dose-limiting toxicities were reported and the maximum tolerated dose of cusatuzumab was not reached. Importantly, cusatuzumab treatment substantially reduced LSCs and triggered gene signatures related to myeloid differentiation and apoptosis.

Phase 2 study of efgartigimod, a novel FcRn antagonist, in adult patients with primary immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is an acquired autoimmune bleeding disorder, characterized by a low platelet count (<100 × 109 /L) in the absence of other causes associated with thrombocytopenia. In most patients, IgG autoantibodies directed against platelet receptors can be detected. They accelerate platelet clearance and destruction, inhibit platelet production, and impair platelet function, resulting in increased risk of bleeding and impaired quality of life. Efgartigimod is a human IgG1 antibody Fc-fragment, a natural ligand of the neonatal Fc receptor (FcRn), engineered for increased affinity to FcRn, while preserving its characteristic pH-dependent binding. Efgartigimod blocks FcRn, preventing IgG recycling, and causing targeted IgG degradation. In this Phase 2 study, 38 patients were randomized 1:1:1 to receive four weekly intravenous infusions of either placebo (N = 12) or efgartigimod at a dose of 5 mg/kg (N = 13) or 10 mg/kg (N = 13). This short treatment cycle of efgartigimod in patients with ITP, predominantly refractory to previous lines of therapy, was shown to be well tolerated, and demonstrated a favorable safety profile consistent with Phase 1 data. Efgartigimod induced a rapid reduction of total IgG levels (up to 63.7% mean change from baseline), which was associated with clinically relevant increases in platelet counts (46% patients on efgartigimod vs 25% on placebo achieved a platelet count of ≥50 × 109 /L on at least two occasions, and 38% vs 0% achieved ≥50 × 109 /L for at least 10 cumulative days), and a reduced proportion of patients with bleeding. Taken together, these data warrant further evaluation of FcRn antagonism as a novel therapeutic approach in ITP.

Monotherapy With Anti-CD70 Antibody Causes Long-Term Mouse Cardiac Allograft Acceptance With Induction of Tolerogenic Dendritic Cells.

Allograft rejection has been an obstacle for the long-term survival of patients. CD70, a tumor necrosis factor (TNF) family member critically expressed on antigen-presenting cells and strongly but transiently up-regulated during lymphocyte activation, represents an important co-stimulatory molecule that induces effective T cell responses. We used a mouse heterotopic cardiac transplantation model to evaluate the effects of monotherapy with the antibody targeting mouse CD70 (FR70) on transplantation tolerance and its immunoregulatory activity. FR70-treated C3H recipient mice permanently accepted B6 fully mismatched cardiac allografts. Consistent with the graft survival, the infiltration of CD8+ T cells in the graft was reduced, dendritic cells were differentiated into a tolerogenic status, and the number of regulatory T cells was elevated both in the graft and the recipient's spleen. In addition, naïve C3H given an adoptive transfer of spleen cells from the primary recipients with FR70 treatment accepted a heart graft from a matching B6 donor but not third-party BALB/c mice.　Our findings show that treatment with FR70 induced regulatory cells and inhibited cytotoxic T cell proliferation, which led to long-term acceptance of mouse cardiac allografts. These findings highlight the potential role of anti-CD70 antibodies as a clinically effective treatment for allograft rejection.

Screening a Broad Range of Solid and Haematological Tumour Types for CD70 Expression Using a Uniform IHC Methodology as Potential Patient Stratification Method.

The constitutive expression of CD70 has been described in various haematological and solid tumour types. In addition, the co-expression of its receptor in tumours has been demonstrated, mediating tumour cell proliferation. Although CD70 expression is a prerequisite to enrol patients in solid tumour clinical trials using anti-CD70 immunotherapy, there is currently no standardised test to evaluate CD70 expression. These differences in immunohistochemistry (IHC) protocols make it challenging to compare the expression levels that were obtained in different studies, pointing out the need for one uniform methodology. In this retrospective study, over 600 tumour samples from different solid and haematological malignancies were analysed while using one validated IHC method. CD70 and CD27 expression was demonstrated in a broad range of tumour types. In solid tumours, 43% demonstrated CD70 positivity with the highest degree in renal cell carcinoma (79.5%). Kaposi sarcoma showed no CD70 expression on the tumour cells. In lymphoma samples, 58% demonstrated CD70 positivity. Moreover, the co-expression of CD70 and CD27 was observed in 39% of lymphoma samples. These findings highlight the need to further explore anti-CD70 therapies in a broad range of CD70 expressing tumour types and in doing so, implementing one standardised protocol to define CD70 overexpression to use it as a diagnostic tool.

VHH-Photosensitizer Conjugates for Targeted Photodynamic Therapy of Met-Overexpressing Tumor Cells.

Photodynamic therapy (PDT) is an approach that kills (cancer) cells by the local production of toxic reactive oxygen species upon the local illumination of a photosensitizer (PS). The specificity of PDT has been further enhanced by the development of a new water-soluble PS and by the specific delivery of PS via conjugation to tumor-targeting antibodies. To improve tissue penetration and shorten photosensitivity, we have recently introduced nanobodies, also known as VHH (variable domains from the heavy chain of llama heavy chain antibodies), for targeted PDT of cancer cells overexpressing the epidermal growth factor receptor (EGFR). Overexpression and activation of another cancer-related receptor, the hepatocyte growth factor receptor (HGFR, c-Met or Met) is also involved in the progression and metastasis of a large variety of malignancies. In this study we evaluate whether anti-Met VHHs conjugated to PS can also serve as a biopharmaceutical for targeted PDT. VHHs targeting the SEMA (semaphorin-like) subdomain of Met were provided with a C-terminal tag that allowed both straightforward purification from yeast supernatant and directional conjugation to the PS IRDye700DX using maleimide chemistry. The generated anti-Met VHH-PS showed nanomolar binding affinity and, upon illumination, specifically killed MKN45 cells with nanomolar potency. This study shows that Met can also serve as a membrane target for targeted PDT.