Transferrin receptor-binding blood-brain barrier shuttle enhances brain delivery and plaque-clearing efficacy of a therapeutic anti-Aβ antibody

Background: Tumor-associated macrophages (TAMs) are the major part of the tumor microenvironment 1, which are classified as two groups - the tumor-suppressing M1 type and the tumor-supporting M2 type 2. Thus, TAMs could act as a double-edged sword. Reprogramming TAMs from M2 to M1 type would improve the efficacy of anti-tumor therapies 3. TAMs may influence the efficacy of therapeutic anti-tumor antibodies. One of the modes of action of therapeutic antibodies is an antibody-dependent cellular phagocytosis (ADCP) 1. However, it is still controversial whether the status of TAMs influences ADCP response 4,5. Resiquimod (R848) is an agonist for Toll-like receptor 7 and 8 (TLR7/8) that can reprogram TAMs from M2 to M1 6. In this study, we evaluated whether R848-induced M1 type TAMs could enhance ADCP response. Therefore, we encapsulated R848 into liposomes, intending to achieve TAM-specific R848 delivery, TAMs re-education, ADCP improvement, and enhancement of therapeutic efficacy of antibodies. Methods: For ADCP assay, mouse bone marrow derived macrophages (BMDM) were differentiated into M1 and M2 as TAM models. Phagocytosis of EGFP-expressing cancer cells (Raji, A431, and SKBR3) by TAMs was evaluated by FACS. For TAM re-education, M2 TAMs were incubated with R848. Expression level of macrophage markers (CD80 and CD206) was measured by FACS. Biodistribution of liposomal R848 (R848-LPs) in WiDr cell-bearing nude mice was evaluated by in vivo imaging and FACS. In vivo TAM re-education ability, ADCP enhancement ability, and tumor growth inhibition ability of R848-LPs combined with therapeutic antibody were investigated in WiDr cell-bearing nude mice. Results: In vitro ADCP assay showed that M1 macrophages elicited the more efficient ADCP response than M2 macrophages towards Raji, A431, and SKBR3 using Rituximab (α-CD20 antibody), α-EGFR antibody (528), and Trastuzumab (α-HER2 antibody), respectively. Upon treatment with R848-LPs, macrophages showed an M1 polarization and an enhanced ADCP response in vitro. In vivo imaging and FACS analysis showed that R848-LPs rapidly accumulated in TAMs at tumor site. R848-LP treatment efficiently enhanced the ADCP response towards WiDr using α-EGFR antibody (528). By the treatment of R848-LPs, a significant increased ratio of CD80/CD206 was observed. WiDr-bearing nude mice treated with R848-LP combined with α-EGFR antibody (528) suppressed tumor growth compared with control groups (non-treatment and α-EGFR antibody without R848-LPs). Conclusions: Liposomal R848 is a potential TAM re-education agent, which could significantly enhance the therapeutic effect of antibodies by improving ADCP. Keywords: tumor-associated macrophage; therapeutic antibodies; antibody-dependent cellular phagocytosis; Resiquimod; liposomes

Therapeutic antibodies – natural and pathological barriers and strategies to overcome them

BackgroundChemotherapeutic drugs and newly developed therapeutic monoclonal antibodies are adequately delivered to most solid and systemic tumors. However, drug delivery into primary brain tumors and metastases is impeded by the blood-brain tumor barrier (BTB), significantly limiting drug use in brain cancer treatment.Methodology/Principal FindingsWe examined the effect of phosphodiesterase 5 (PDE5) inhibitors in nude mice on drug delivery to intracranially implanted human lung and breast tumors as the most common primary tumors forming brain metastases, and studied underlying mechanisms of drug transport. In vitro assays demonstrated that PDE5 inhibitors enhanced the uptake of [14C]dextran and trastuzumab (Herceptin®, a humanized monoclonal antibody against HER2/neu) by cultured mouse brain endothelial cells (MBEC). The mechanism of drug delivery was examined using inhibitors for caveolae-mediated endocytosis, macropinocytosis and coated pit/clathrin endocytosis. Inhibitor analysis strongly implicated caveolae and macropinocytosis endocytic pathways involvement in the PDE5 inhibitor-enhanced Herceptin uptake by MBEC. Oral administration of PDE5 inhibitor, vardenafil, to mice with HER2-positive intracranial lung tumors led to an increased tumor permeability to high molecular weight [14C]dextran (2.6-fold increase) and to Herceptin (2-fold increase). Survival time of intracranial lung cancer-bearing mice treated with Herceptin in combination with vardenafil was significantly increased as compared to the untreated, vardenafil- or Herceptin-treated mice (p<0.01). Log-rank survival analysis of mice bearing HER2-positive intracranial breast tumor also showed a significant survival increase (p<0.02) in the group treated with Herceptin plus vardenafil as compared to other groups. However, vardenafil did not exert any beneficial effect on survival of mice bearing intracranial breast tumor with low HER2 expression and co-treated with Herceptin (p>0.05).Conclusions/SignificanceThese findings suggest that PDE5 inhibitors may effectively modulate BTB permeability, and enhance delivery and therapeutic efficacy of monoclonal antibodies in hard-to-treat brain metastases from different primary tumors that had metastasized to the brain.

Purpose – Antibodies of the human (hu) Immunoglobulin G (IgG) isotype are used as therapeutics for patients with cancer, rheumatoid arthritis, asthma, and other diseases. Often, these therapeutic huIgG antibodies mediate effects by binding to human Fc gamma receptors (FcγRs) expressed on various cells of the patient’s immune system. Three classes of huFcγRs comprising a total of six receptors are known in humans, namely FcγRIa (CD64), FcγRIIa/b/c (CD32a/b/c), and FcγRIIIa/b (CD16a/b). FcγR-mediated effector functions range from desired depletion of tumor cells via antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis, to unwanted toxic effects by exaggerated cytokine release, thrombosis, and infusion reactions. These functions depend on the FcγR, the binding strength, and the involved immune cells. Prior to human use, the safety and efficacy of therapeutics have to be demonstrated in animal studies where human antibodies interact with the immune system of the selected species. The Gottingen minipig is highly suitable for such mandatory preclinical studies. However, the relevance of such studies for assessing the safety and efficacy of therapeutic antibodies is limited due to unknown characteristics of porcine (po)FcγRs. Therefore, this thesis aims to characterize the poFcγRs, focusing on the expression on immune cells of the minipig and the binding to huIgG. Methods – To study the set of poFcγRs in minipigs, we performed a detailed genome analysis of the locus coding for most FcγRs by polymerase chain reaction (PCR) and manual assembly of existing sequences. We used single cell ribonucleic acid (RNA) sequencing to determine the transcription, and flow cytometry to show the expression of different poFcγRs on various cells within blood, lymph node, and spleen. Cloning and expression of all poFcγRs as soluble proteins enabled the binding assessment of monomeric, as well as immune complexed huIgG1 therapeutic antibodies to poFcγRs by surface plasmon resonance (SPR; Biacore). Furthermore, we investigated the binding of monomeric antibodies and immune complexes to FcγR-expressing cell lines and immune cells of the minipig by flow cytometry. Results – We used genome analysis to identify the missing poFcγRIIa and to map the gene coding for the known poFcγRIIIa, which had not been annotated to date. The genomic organization of poFcγRs resembles that of most mammals except humans, who have two additional genes coding for huFcγRIIc and IIIb. In general, the distribution of FcγRs on immune cells and the binding properties to free- and immune-complexed huIgG1, both prerequisites for effector functions mediated by huIgG1, are similar in minipigs and humans. However, we observed several key differences which may affect the use of minipigs in preclinical studies with therapeutic huIgG1 antibodies. Firstly, the binding of huIgG1 to FcγRIIa, which is expressed on blood platelets, was stronger in minipigs (poFcγRIIa) compared to humans (huFcγRIIa). Despite this, the minipig could be a valuable model to study IgG-mediated platelet activation, aggregation, and thrombosis. Secondly, for the inhibitory poFcγRIIb, we observed stronger binding versus huFcγRIIb. In humans, FcγRIIb regulates the immune response and is expressed on B cells, dendritic cells, and tissue monocytes. In contrast, we reported expression of poFcγRIIb on blood monocytes in minipigs. We suggest that anti-inflammatory effects with therapeutic huIgG1 antibodies could be stronger in minipigs than in humans due to the divergent expression and the stronger binding to the inhibitory poFcγRIIb. Lastly, we observed a lack of binding of huIgG1 to poFcγRIIIa. In humans, cytotoxic huIgG1 antibodies mediate ADCC via binding to huFcγRIIIa expressed on natural killer (NK) cells and on a subset of monocytes in the blood. The lacking binding of huIgG1 to poFcγRIIIa excludes NK-mediated ADCC and additionally restricts functions of monocytes, thus limiting studies with certain huIgG1 therapeutics. However, we reported binding of endogenous poIgG1 enabling effector functions in tumor vaccination or infection studies. Conclusion – The results compiled in this thesis generally recommend the use of minipigs for the assessment of therapeutic huIgG1 antibodies. However, the limitations of this animal model regarding differential binding of huIgG1 to poFcγRs and their expression pattern on immune cells in comparison to the human have to be considered. Therefore, functional studies are recommended to further assess the translatability of FcγR-mediated effector functions with various therapeutic antibodies from the minipig to the human. Nevertheless, this work delivers a foundation for species selection and allows the interpretation of results from preclinical safety and efficacy studies with Gottingen minipigs.

Therapeutic antibody drug development is a rapidly growing sector of the pharmaceutical industry. However, antibody drug development for the brain is a technical challenge, and therapeutic antibodies for the central nervous system account for ∼3% of all such agents. The principal obstacle to antibody drug development for brain or spinal cord is the lack of transport of large molecule biologics across the blood-brain barrier (BBB). Therapeutic antibodies can be made transportable through the blood-brain barrier by the re-engineering of the therapeutic antibody as a BBB-penetrating bispecific antibody (BSA). One arm of the BSA is the therapeutic antibody and the other arm of the BSA is a transporting antibody. The transporting antibody targets an exofacial epitope on a BBB receptor, and this enables receptor-mediated transcytosis (RMT) of the BSA across the BBB. Following BBB transport, the therapeutic antibody then engages the target receptor in brain. RMT systems at the BBB that are potential conduits to the brain include the insulin receptor (IR), the transferrin receptor (TfR), the insulin-like growth factor receptor (IGFR) and the leptin receptor. Therapeutic antibodies have been re-engineered as BSAs that target the insulin receptor, TfR, or IGFR RMT systems at the BBB for the treatment of Alzheimer’s disease and Parkinson’s disease.

Parathyroid Hormone-related Peptide (PTHrP)is a potent regulator of bone turnover and is thought to play a major role in the progression of skeletal metastasis through its activation of growth factors in the bone microenvironment. Our previous studies using genetically engineered mouse models in which PTHrP was ablated in the mammary epithelium have demonstrated that PTHrP also plays a direct role on tumor progression by enhancing tumor initiation, growth and metastasis outside the skeleton. We therefore proposed a novel mechanism of PTHrP driven bone metastasis by which PTHrP controls both the seed (tumor cells) and the soil (the bone microenvironment). We tested our hypothesis in triple negative breast cancer cell line (TNBC)models which are not responsive to either tamoxifen or Herceptin. Monoclonal antibodies against PTHrP were generated with strong in vitro anti-proliferative and anti-invasive potency in two human PTHrP expressing cell lines: the widely used TNBC MDAMB231 cell line and a patient derived TNBC cell line. Ablation of PTHrP or treatment with anti-PTHrP monoclonal antibodies induced major changes in the phenotype of these cells as determined by flow cytometry with stem cells and EMT markers, invasion assays and mammosphere assays indicating a reversal of their metastatic/invasive phenotype. Next we transplanted these cell lines into 8 weeks old female athymic nude mice to examine the efficacy of the therapeutic monoclonal antibodies on the progression of established bone metastasis.104 tumor cells were injected intra-tibially and animals examined at timed intervals(14, 21, 28, 25 and 42 days) with X-rays and high resolution CTs.At 14 days animals were administered intra-peritoneally with 200 micrograms of therapeutic anti-PTHrP antibodies or non-immune IgG(10 animals per group) and every 3 days thereafter til sacrifice(42 days). At sacrifice PET scan was performed to measure tumor volume and activity and bone were collected for histomorphometry analysis of tumor size and bone turnover activity. A significant reduction in bone lesions (P&amp;lt;0.05) was observed over time by imaging in animals treated with the therapeutic antibodies. At sacrifice PET scan indicates a strong reduction in tumor activity in treated animals (P&amp;lt;0.05). Histomorphometry analysis showed greater than 50% reduction in tumor volume associated with a significant inhibition of bone turnover in treated animals (P&amp;lt;0.05). In summary our data demonstrate that, in two TNBC models, PTHrP blockade can reprogram cancer cells to a reduced tumorigenicity and block the progression of established bone metastasis by targeting both the tumor cell and the bone microenvironment. Our data lend support to the “seed and soil” hypothesis proposed by Paget and the rational of anti-PTHrP therapeutic strategies alone or in combination with bone targeting agents in order to eradicate the development of skeletal metastasis. Citation Format: Jiarong Li, Anne Camirand, Mahvash Zakikhani, Karine Sellin, Richard Kremer. Therapeutic antibodies against parathyroid hormone-related peptide (PTHrP) inhibit the growth of established skeletal metastasis in mice transplanted with human triple negative breast cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 520.

Clinical efficacy and management of monoclonal antibodies targeting CD38 and SLAMF7 in multiple myeloma

Glycosylation of the Fc region of IgG has a profound impact on the safety and clinical efficacy of therapeutic antibodies. While the biantennary complex-type oligosaccharide attached to Asn297 of the Fc is essential for antibody effector functions, fucose and outer-arm sugars attached to the core heptasaccharide that generate structural heterogeneity (glycoforms) exhibit unique biological activities. Hence, efficient and quantitative glycan analysis techniques have been increasingly important for the development and quality control of therapeutic antibodies, and glycan profiles of the Fc are recognized as critical quality attributes. In the past decade our understanding of the influence of glycosylation on the structure/function of IgG-Fc has grown rapidly through X-ray crystallographic and nuclear magnetic resonance studies, which provides possibilities for the design of novel antibody therapeutics. Furthermore, the chemoenzymatic glycoengineering approach using endoglycosidase-based glycosynthases may facilitate the development of homogeneous IgG glycoforms with desirable functionality as next-generation therapeutic antibodies. Thus, the Fc glycans are fertile ground for the improvement of the safety, functionality, and efficacy of therapeutic IgG antibodies in the era of precision medicine.

Antibody therapeutics are limited in treating brain diseases due to poor blood-brain barrier (BBB) penetration. We have discovered that poly 2-methacryloyloxyethyl phosphorylcholine (PMPC), a biocompatible polymer, effectively facilitates BBB penetration via receptor-mediated transcytosis and have developed a PMPC-shell-based platform for brain delivery of therapeutic antibodies, termed nanocapsule. Yet, the platform results in functional loss of antibodies due to epitope masking by the PMPC polymer network, which necessitates the incorporation of a targeting moiety and degradable crosslinker to enable on-site antibody release. In this study, we developed a novel platform based on site-oriented conjugation of PMPC to the antibody, allowing it to maintain key functionalities of the original antibody. With an optimized PMPC chain length, the PMPC-antibody conjugate exhibited enhanced brain delivery while retaining epitope recognition, cellular internalization, and antibody-dependent cellular phagocytic activity. This simple formula incorporates only the antibody and PMPC without requiring additional components, thereby addressing the issues of the nanocapsule platform and paving the way for PMPC-based brain delivery strategies for antibodies.

An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo

An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo

The efficacy of antibody-based therapeutics depends on successful drug delivery into solid tumors; therefore, there is a clinical need to measure intratumoral antibody distribution. This study aims to develop and validate an imaging and computation platform to directly quantify and predict antibody delivery into human head and neck cancers in a clinical study. Twenty-four patients received systemic infusion of a near-infrared fluorescence-labeled therapeutic antibody followed by surgical tumor resection. A computational platform was developed to quantify the extent of heterogeneity of intratumoral antibody distribution. Both univariate and multivariate regression analyses were used to select the most predictive tumor biological factors for antibody delivery. Quantitative image features from the pretreatment MRI were extracted and correlated with fluorescence imaging of antibody delivery. This study not only confirmed heterogeneous intratumoral antibody distribution in-line with many preclinical reports, but also quantified the extent of interpatient, intertumor, and intratumor heterogeneity of antibody delivery. This study demonstrated the strong predictive value of tumor size for intratumoral antibody accumulation and its significant impact on antibody distribution in both primary tumor and lymph node metastasis. Furthermore, this study established the feasibility of using contrast-enhanced MRI to predict antibody delivery. This study provides a clinically translatable platform to measure antibody delivery into solid tumors and yields valuable insight into clinically relevant antibody tumor penetration, with implications in the selection of patients amenable to antibody therapy and the design of more effective dosing strategies.

&lt;div&gt;AbstractPurpose:&lt;p&gt;The efficacy of antibody-based therapeutics depends on successful drug delivery into solid tumors; therefore, there is a clinical need to measure intratumoral antibody distribution. This study aims to develop and validate an imaging and computation platform to directly quantify and predict antibody delivery into human head and neck cancers in a clinical study.&lt;/p&gt;Experimental Design:&lt;p&gt;Twenty-four patients received systemic infusion of a near-infrared fluorescence-labeled therapeutic antibody followed by surgical tumor resection. A computational platform was developed to quantify the extent of heterogeneity of intratumoral antibody distribution. Both univariate and multivariate regression analyses were used to select the most predictive tumor biological factors for antibody delivery. Quantitative image features from the pretreatment MRI were extracted and correlated with fluorescence imaging of antibody delivery.&lt;/p&gt;Results:&lt;p&gt;This study not only confirmed heterogeneous intratumoral antibody distribution in-line with many preclinical reports, but also quantified the extent of interpatient, intertumor, and intratumor heterogeneity of antibody delivery. This study demonstrated the strong predictive value of tumor size for intratumoral antibody accumulation and its significant impact on antibody distribution in both primary tumor and lymph node metastasis. Furthermore, this study established the feasibility of using contrast-enhanced MRI to predict antibody delivery.&lt;/p&gt;Conclusions:&lt;p&gt;This study provides a clinically translatable platform to measure antibody delivery into solid tumors and yields valuable insight into clinically relevant antibody tumor penetration, with implications in the selection of patients amenable to antibody therapy and the design of more effective dosing strategies.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;The efficacy of antibody-based therapeutics depends on successful drug delivery into solid tumors; therefore, there is a clinical need to measure intratumoral antibody distribution. This study aims to develop and validate an imaging and computation platform to directly quantify and predict antibody delivery into human head and neck cancers in a clinical study.&lt;/p&gt;Experimental Design:&lt;p&gt;Twenty-four patients received systemic infusion of a near-infrared fluorescence-labeled therapeutic antibody followed by surgical tumor resection. A computational platform was developed to quantify the extent of heterogeneity of intratumoral antibody distribution. Both univariate and multivariate regression analyses were used to select the most predictive tumor biological factors for antibody delivery. Quantitative image features from the pretreatment MRI were extracted and correlated with fluorescence imaging of antibody delivery.&lt;/p&gt;Results:&lt;p&gt;This study not only confirmed heterogeneous intratumoral antibody distribution in-line with many preclinical reports, but also quantified the extent of interpatient, intertumor, and intratumor heterogeneity of antibody delivery. This study demonstrated the strong predictive value of tumor size for intratumoral antibody accumulation and its significant impact on antibody distribution in both primary tumor and lymph node metastasis. Furthermore, this study established the feasibility of using contrast-enhanced MRI to predict antibody delivery.&lt;/p&gt;Conclusions:&lt;p&gt;This study provides a clinically translatable platform to measure antibody delivery into solid tumors and yields valuable insight into clinically relevant antibody tumor penetration, with implications in the selection of patients amenable to antibody therapy and the design of more effective dosing strategies.&lt;/p&gt;&lt;/div&gt;

Passive administration of broadly neutralizing anti-influenza monoclonal antibodies (mAbs) before or after virus infection can prevent or alleviate disease. Unlike seasonal influenza, infection with zoonotic avian influenza viruses can lead to acute respiratory distress syndrome and high mortality in humans. Respiratory tract-targeting antibody delivery appears to be more clinically relevant and effective for zoonotic influenza treatment. In this study, the efficacy of an anti-H7N9 murine mAb 4B7 and its humanized form (chi4B7) against H7 subtype influenza viruses administered through the intranasal route was investigated in mice. 4B7 recognizes critical residues in the vestigial esterase domain and receptor-binding sites in the hemagglutinin of H7N9 virus. The antibody had cross-H7 binding, hemagglutination inhibition, and neutralizing activities. In particular, the dose of 4B7 required for prophylactic protection against H7N9 infection was significantly reduced in mice treated locally (intranasal) compared with those treated systemically (intraperitoneal). Intranasal delivery of the antibody also enhanced therapeutic efficacy against H7N9 infection compared to intraperitoneal administration. Chi4B7 generated by grafting the variable regions onto the human IgG1 backbone sustained cross-reactivity with different H7 viruses of the parental murine antibody. Airway-delivered chi4B7 provided broad prophylactic and therapeutic protection against divergent H7 viruses in mice. Moreover, intranasal administration of chi4B7 had a long effective prophylaxis window against H7N9 infection. Our results suggest that airway delivery of the humanized anti-H7 antibody is a favorable approach for broad-spectrum prophylaxis and therapy against the H7 subtype influenza.IMPORTANCEInfection of zoonotic H7 avian influenza viruses can cause severe respiratory symptoms and high mortality in humans. Monoclonal antibody administration is an effective approach for treatment of zoonotic influenza infection, while systematic routes of antibody administration (typically intravenous infusion) have several shortcomings. However, there are no approved anti-H7 antibody therapies, and the efficacy of antibodies administered through the airway route against H7 viruses has not been fully investigated. Herein, we report a murine broadly neutralizing monoclonal antibody against divergent H7 viruses and reveal that intranasal administration enhanced prophylactic and therapeutic efficacy of this antibody against H7N9 virus compared to systemic administration. Airway delivery of the humanized antibody conferred broad protection against diverse strains of H7 virus in mice. Our study presents new candidates of broad antiviral agents against H7 avian influenza viruses and highlights airway delivery as a more potent manner of administering antibodies for clinical treatment of influenza.