Transcriptional signature of durable effector T cells elicited by a replication defective HCMV vaccine.

Human cytomegalovirus (HCMV) is a leading infectious cause of birth defects and the most common opportunistic infection that causes life-threatening diseases post-transplantation; however, an effective vaccine remains elusive. V160 is a live-attenuated replication defective HCMV vaccine that showed a 42.4% efficacy against primary HCMV infection among seronegative women in a phase 2b clinical trial. Here, we integrated the multicolor flow cytometry, longitudinal T cell receptor (TCR) sequencing, and single-cell RNA/TCR sequencing approaches to characterize the magnitude, phenotype, and functional quality of human T cell responses to V160. We demonstrated that V160 de novo induces IE-1 and pp65 specific durable polyfunctional effector CD8 T cells that are comparable to those induced by natural HCMV infection. We identified a variety of V160-responsive T cell clones which exhibit distinctive "transient" and "durable" expansion kinetics, and revealed a transcriptional signature that marks durable CD8 T cells post-vaccination. Our study enhances the understanding of human T-cell immune responses to V160 vaccination.

Fc gamma receptors promote antibody-induced LILRB4 internalization and immune regulation of monocytic AML.

The immune checkpoint leukocyte immunoglobulin-like receptor B4 (LILRB4) is found specifically on the cell surface of acute monocytic leukemia (monocytic AML), an aggressive and common subtype of AML. We have developed a humanized monoclonal IgG1 LILRB4-blocking antibody (h128-3), which improved immune regulation but reduced cell surface expression of LILRB4 in monocytic AML models by 40-60%. Interestingly, most of this effect was neutralized by mutation of the Fc region of the antibody (h128-3/N297A), which prevents interaction with Fc gamma receptors (FcγRs). This suggested that there is FcγR-dependent antigenic modulation underlying h128-3's effects, a mechanism known to alter the function of antibodies targeting B-cell malignancies. We disrupted the Fc-FcγR interaction pharmacologically and with stable CRISPR-Cas9-mediated genetic knockout of FcγRs in monocytic AML cell lines to investigate the role of FcγR-dependent antigenic modulation in the regulation of LILRB4 by h128-3. When FcγRI is inhibited or removed from the surface of monocytic AML cells, h128-3 cannot optimally perform its blocking function, resulting in activation of the LILRB4 inhibitory receptor and leading to a 15-25% decrease in T-cell-mediated cytotoxicity in vitro. In the absence of FcγRI, scaffolding by FcγRIIa allows h128-3 to maintain LILRB4-blocking function. Here we define a FcγR-dependent antigenic modulation mechanism underlying the function of an immunoreceptor blocking antibody for the first time in myeloid malignancy. This research will facilitate the development of safe, precision-targeted antibody therapeutics in myeloid malignancies with greater potency and efficacy.

LILRB3 Supports Immunosuppressive Activity of Myeloid Cells and Tumor Development.

The existing T cell-centered immune checkpoint blockade therapies have been successful in treating some but not all patients with cancer. Immunosuppressive myeloid cells, including myeloid-derived suppressor cells (MDSC), that inhibit antitumor immunity and support multiple steps of tumor development are recognized as one of the major obstacles in cancer treatment. Leukocyte Ig-like receptor subfamily B3 (LILRB3), an immune inhibitory receptor containing tyrosine-based inhibitory motifs (ITIM), is expressed solely on myeloid cells. However, it is unknown whether LILRB3 is a critical checkpoint receptor in regulating the activity of immunosuppressive myeloid cells, and whether LILRB3 signaling can be blocked to activate the immune system to treat solid tumors. Here, we report that galectin-4 and galectin-7 induce activation of LILRB3 and that LILRB3 is functionally expressed on immunosuppressive myeloid cells. In some samples from patients with solid cancers, blockade of LILRB3 signaling by an antagonistic antibody inhibited the activity of immunosuppressive myeloid cells. Anti-LILRB3 also impeded tumor development in myeloid-specific LILRB3 transgenic mice through a T cell-dependent manner. LILRB3 blockade may prove to be a novel approach for immunotherapy of solid cancers.

An ACE2 decamer viral trap as a durable intervention solution for current and future SARS-CoV.

The capacity of SARS-CoV-2 to evolve poses challenges to conventional prevention and treatment options such as vaccination and monoclonal antibodies, as they rely on viral receptor binding domain (RBD) sequences from previous strains. Additionally, animal CoVs, especially those of the SARS family, are now appreciated as a constant pandemic threat. We present here a new antiviral approach featuring inhalation delivery of a recombinant viral trap composed of ten copies of angiotensin-converting enzyme 2 (ACE2) fused to the IgM Fc. This ACE2 decamer viral trap is designed to inhibit SARS-CoV-2 entry function, regardless of viral RBD sequence variations as shown by its high neutralization potency against all known SARS-CoV-2 variants, including Omicron BQ.1, BQ.1.1, XBB.1 and XBB.1.5. In addition, it demonstrates potency against SARS-CoV-1, human NL63, as well as bat and pangolin CoVs. The multivalent trap is effective in both prophylactic and therapeutic settings since a single intranasal dosing confers protection in human ACE2 transgenic mice against viral challenges. Lastly, this molecule is stable at ambient temperature for more than twelve weeks and can sustain physical stress from aerosolization. These results demonstrate the potential of a decameric ACE2 viral trap as an inhalation solution for ACE2-dependent coronaviruses of current and future pandemic concerns.

Hyperleptinemia contributes to antipsychotic drug-associated obesity and metabolic disorders.

Despite their high degree of effectiveness in the management of psychiatric conditions, exposure to antipsychotic drugs, including olanzapine and risperidone, is frequently associated with substantial weight gain and the development of diabetes. Even before weight gain, a rapid rise in circulating leptin concentrations can be observed in most patients taking antipsychotic drugs. To date, the contribution of this hyperleptinemia to weight gain and metabolic deterioration has not been defined. Here, with an established mouse model that recapitulates antipsychotic drug-induced obesity and insulin resistance, we not only confirm that hyperleptinemia occurs before weight gain but also demonstrate that hyperleptinemia contributes directly to the development of obesity and associated metabolic disorders. By suppressing the rise in leptin through the use of a monoclonal leptin-neutralizing antibody, we effectively prevented weight gain, restored glucose tolerance, and preserved adipose tissue and liver function in antipsychotic drug-treated mice. Mechanistically, suppressing excess leptin resolved local tissue and systemic inflammation typically associated with antipsychotic drug treatment. We conclude that hyperleptinemia is a key contributor to antipsychotic drug-associated weight gain and metabolic deterioration. Leptin suppression may be an effective approach to reducing the undesirable side effects of antipsychotic drugs.

Vaccination with a replication-defective cytomegalovirus vaccine elicits a glycoprotein B-specific monoclonal antibody repertoire distinct from natural infection.

Human Cytomegalovirus (HCMV) is the leading infectious congenital infection globally and the most common viral infection in transplant recipients, therefore identifying a vaccine for HCMV is a top priority. Humoral immunity is a correlate of protection for HCMV infection. The most effective vaccine tested to date, which achieved 50% reduction in acquisition of HCMV, was comprised of the glycoprotein B protein given with an oil-in-water emulsion adjuvant MF59. We characterize gB-specific monoclonal antibodies isolated from individuals vaccinated with a disabled infectious single cycle (DISC) CMV vaccine, V160, and compare these to the gB-specific monoclonal antibody repertoire isolated from naturally-infected individuals. We find that vaccination with V160 resulted in gB-specific antibodies that bound homogenously to gB expressed on the surface of a cell in contrast to antibodies isolated from natural infection which variably bound to cell-associated gB. Vaccination resulted in a similar breadth of gB-specific antibodies, with binding profile to gB genotypes 1-5 comparable to that of natural infection. Few gB-specific neutralizing antibodies were isolated from V160 vaccinees and fewer antibodies had identifiable gB antigenic domain specificity compared to that of naturally-infected individuals. We also show that glycosylation of gB residue N73 may shield binding of gB-specific antibodies.

A highly selective humanized DDR1 mAb reverses immune exclusion by disrupting collagen fiber alignment in breast cancer.

BACKGROUND: Immune exclusion (IE) where tumors deter the infiltration of immune cells into the tumor microenvironment has emerged as a key mechanism underlying immunotherapy resistance. We recently reported a novel role of discoidin domain-containing receptor 1 (DDR1) in promoting IE in breast cancer and validated its critical role in IE using neutralizing rabbit monoclonal antibodies (mAbs) in multiple mouse tumor models. METHODS: To develop a DDR1-targeting mAb as a potential cancer therapeutic, we humanized mAb9 with a complementarity-determining region grafting strategy. The humanized antibody named PRTH-101 is currently being tested in a Phase 1 clinical trial. We determined the binding epitope of PRTH-101 from the crystal structure of the complex between DDR1 extracellular domain (ECD) and the PRTH-101 Fab fragment with 3.15 Å resolution. We revealed the underlying mechanisms of action of PRTH-101 using both cell culture assays and in vivo study in a mouse tumor model. RESULTS: PRTH-101 has subnanomolar affinity to DDR1 and potent antitumor efficacy similar to the parental rabbit mAb after humanization. Structural information illustrated that PRTH-101 interacts with the discoidin (DS)-like domain, but not the collagen-binding DS domain of DDR1. Mechanistically, we showed that PRTH-101 inhibited DDR1 phosphorylation, decreased collagen-mediated cell attachment, and significantly blocked DDR1 shedding from the cell surface. Treatment of tumor-bearing mice with PRTH-101 in vivo disrupted collagen fiber alignment (a physical barrier) in the tumor extracellular matrix (ECM) and enhanced CD8+ T cell infiltration in tumors. CONCLUSIONS: This study not only paves a pathway for the development of PRTH-101 as a cancer therapeutic, but also sheds light on a new therapeutic strategy to modulate collagen alignment in the tumor ECM for enhancing antitumor immunity.

Overcoming adaptive resistance to anti-VEGF therapy by targeting CD5L.

Antiangiogenic treatment targeting the vascular endothelial growth factor (VEGF) pathway is a powerful tool to combat tumor growth and progression; however, drug resistance frequently emerges. We identify CD5L (CD5 antigen-like precursor) as an important gene upregulated in response to antiangiogenic therapy leading to the emergence of adaptive resistance. By using both an RNA-aptamer and a monoclonal antibody targeting CD5L, we are able to abate the pro-angiogenic effects of CD5L overexpression in both in vitro and in vivo settings. In addition, we find that increased expression of vascular CD5L in cancer patients is associated with bevacizumab resistance and worse overall survival. These findings implicate CD5L as an important factor in adaptive resistance to antiangiogenic therapy and suggest that modalities to target CD5L have potentially important clinical utility.

Antibody therapies for the treatment of acute myeloid leukemia: exploring current and emerging therapeutic targets.

INTRODUCTION: Acute myeloid leukemia (AML) is the most common and deadly type of leukemia affecting adults. It is typically managed with rounds of non-targeted chemotherapy followed by hematopoietic stem cell transplants, but this is only possible in patients who can tolerate these harsh treatments and many are elderly and frail. With the identification of novel tumor-specific cell surface receptors, there is great conviction that targeted antibody therapies will soon become available for these patients. AREAS COVERED: In this review, we describe the current landscape of known target receptors for monospecific and bispecific antibody-based therapeutics for AML. Here, we characterize each of the receptors and targeted antibody-based therapeutics in development, illustrating the rational design behind each therapeutic compound. We then discuss the bispecific antibodies in development and how they improve immune surveillance of AML. For each therapeutic, we also summarize the available pre-clinical and clinical data, including data from discontinued trials. EXPERT OPINION: One antibody-based therapeutic has already been approved for AML treatment, the CD33-targeting antibody-drug conjugate, gemtuzumab ozogamicin. Many more are currently in pre-clinical and clinical studies. These antibody-based therapeutics can perform tumor-specific, elaborate cytotoxic functions and there is growing confidence they will soon lead to personalized, safe AML treatment options that induce durable remissions.

A novel humanized Chi3l1 blocking antibody attenuates acetaminophen-induced liver injury in mice.

Acetaminophen (APAP) overdose is a leading cause of acute liver injury in the USA. The chitinase 3-like-1 (Chi3l1) protein contributes to APAP-induced liver injury (AILI) by promoting hepatic platelet recruitment. Here, we report the development of a Chi3l1-targeting antibody as a potential therapy for AILI. By immunizing a rabbit successively with the human and mouse Chi3l1 proteins, we isolated cross-reactive monoclonal antibodies (mAbs) from single memory B cells. One of the human and mouse Chi3l1 cross-reactive mAbs was humanized and characterized in both in vitro and in vivo biophysical and biological assays. X-ray crystallographic analysis of the lead antibody C59 in complex with the human Chi3l1 protein revealed that the kappa light contributes to majority of the antibody-antigen interaction; and that C59 binds to the 4α-5ß loop and 4α-helix of Chi3l1, which is a functional epitope and hotspot for the development of Chi3l1 blocking antibodies. We humanized the C59 antibody by complementarity-determining region grafting and kappa chain framework region reverse mutations. The humanized C59 antibody exhibited similar efficacy as the parental rabbit antibody C59 in attenuating AILI in vivo. Our findings validate Chi3l1 as a potential drug target for AILI and provide proof of concept of developing Chi3l1 blocking antibody as a therapy for the treatment of AILI.

Endotrophin neutralization through targeted antibody treatment protects from renal fibrosis in a podocyte ablation model.

OBJECTIVE: Renal fibrosis is a hallmark for chronic kidney disease (CKD), and often leads to end stage renal disease (ESRD). However, limited interventions are available clinically to ameliorate or reverse renal fibrosis. METHODS: Herein, we evaluated whether blockade of endotrophin through neutralizing antibodies protects from renal fibrosis in the podocyte insult model (the "POD-ATTAC" mouse). We determined the therapeutic effects of endotrophin targeted antibody through assessing renal function, renal inflammation and fibrosis at histological and transcriptional levels, and podocyte regeneration. RESULTS: We demonstrated that neutralizing endotrophin antibody treatment significantly ameliorates renal fibrosis at the transcriptional, morphological, and functional levels. In the antibody treatment group, expression of pro-inflammatory and pro-fibrotic genes was significantly reduced, normal renal structures were restored, collagen deposition was decreased, and proteinuria and renal function were improved. We further performed a lineage tracing study confirming that podocytes regenerate as de novo podocytes upon injury and loss, and blockade of endotrophin efficiently enhances podocyte-specific marker expressions. CONCLUSION: Combined, we provide pre-clinical evidence supporting neutralizing endotrophin as a promising therapy for intervening with renal fibrosis in CKD, and potentially in other chronic fibro-inflammatory diseases.

A potent and broad neutralization of SARS-CoV-2 variants of concern by DARPins.

We report the engineering and selection of two synthetic proteins-FSR16m and FSR22-for the possible treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. FSR16m and FSR22 are trimeric proteins composed of DARPin SR16m or SR22 fused with a T4 foldon. Despite selection by a spike protein from a now historical SARS-CoV-2 strain, FSR16m and FSR22 exhibit broad-spectrum neutralization of SARS-CoV-2 strains, inhibiting authentic B.1.351, B.1.617.2 and BA.1.1 viruses, with respective IC50 values of 3.4, 2.2 and 7.4 ng ml-1 for FSR16m. Cryo-EM structures revealed that these DARPins recognize a region of the receptor-binding domain (residues 456, 475, 486, 487 and 489) overlapping a critical portion of the angiotensin-converting enzyme 2 (ACE2)-binding surface. K18-hACE2 transgenic mice inoculated with B.1.617.2 and receiving intranasally administered FSR16m showed less weight loss and 10-100-fold lower viral burden in upper and lower respiratory tracts. The strong and broad neutralization potency makes FSR16m and FSR22 promising candidates for the prevention and treatment of infection by SARS-CoV-2.

Engineering antibody and protein therapeutics to cross the blood-brain barrier.

Diseases in the central nervous system (CNS) are often difficult to treat. Antibody- and protein-based therapeutics hold huge promises in CNS disease treatment. However, proteins are restricted from entering the CNS by the blood-brain barrier (BBB). To achieve enhanced BBB crossing, antibody-based carriers have been developed by utilizing the endogenous macromolecule transportation pathway, known as receptor-mediated transcytosis. In this report, we first provided an overall review on key CNS diseases and the most promising antibody- or protein-based therapeutics approved or in clinical trials. We then reviewed the platforms that are being explored to increase the macromolecule brain entry to combat CNS diseases. Finally, we have analyzed the lessons learned from past experiences and have provided a perspective on the future engineering of novel delivery vehicles for antibody- and protein-based therapies for CNS diseases.

Anti-GRP-R monoclonal antibody antitumor therapy against neuroblastoma.

Standard treatment for patients with high-risk neuroblastoma remains multimodal therapy including chemoradiation, surgical resection, and autologous stem cell rescue. Immunotherapy has demonstrated success in treating many types of cancers; however, its use in pediatric solid tumors has been limited by low tumor mutation burdens. Gastrin-releasing peptide receptor (GRP-R) is overexpressed in numerous malignancies, including poorly-differentiated neuroblastoma. Monoclonal antibodies (mAbs) to GRP-R have yet to be developed but could serve as a potential novel immunotherapy. This preclinical study aims to evaluate the efficacy of a novel GRP-R mAb immunotherapy against neuroblastoma. We established four candidate anti-GRP-R mAbs by screening a single-chain variable fragment (scFv) library. GRP-R mAb-1 demonstrated the highest efficacy with the lowest EC50 at 4.607 ng/ml against GRP-R expressing neuroblastoma cells, blocked the GRP-ligand activation of GRP-R and its downstream PI3K/AKT signaling. This resulted in functional inhibition of cell proliferation and anchorage-independent growth, indicating that mAb-1 has an antagonist inhibitory role on GRP-R. To examine the antibody-dependent cellular cytotoxicity (ADCC) of GRP-R mAb-1 on neuroblastoma, we co-cultured neuroblastoma cells with natural killer (NK) cells versus GRP-R mAb-1 treatment alone. GRP-R mAb-1 mediated ADCC effects on neuroblastoma cells and induced release of IFNγ by NK cells under co-culture conditions in vitro. The cytotoxic effects of mAb-1 were confirmed with the secretion of cytotoxic granzyme B from NK cells and the reduction of mitotic tumor cells in vivo using a murine tumor xenograft model. In summary, GRP-R mAb-1 demonstrated efficacious anti-tumor effects on neuroblastoma cells in preclinical models. Importantly, GRP-R mAb-1 may be an efficacious, novel immunotherapy in the treatment of high-risk neuroblastoma patients.

Blocking LAIR1 signaling in immune cells inhibits tumor development.

The current immune checkpoint blockade therapy has been successful in treating some cancers but not others. New molecular targets and therapeutic approaches of cancer immunology need to be identified. Leukocyte associated immunoglobulin like receptor 1 (LAIR1) is an immune inhibitory receptor expressing on most immune cell types. However, it remains a question whether we can specifically and actively block LAIR1 signaling to activate immune responses for cancer treatment. Here we report the development of specific antagonistic anti-LAIR1 monoclonal antibodies and studied the effects of LAIR1 blockade on the anti-tumor immune functions. The anti-LAIR1 antagonistic antibody stimulated the activities of T cells, natural killer cells, macrophages, and dendritic cells in vitro. The single-cell RNA sequencing analysis of intratumoral immune cells in syngeneic human LAIR1 transgenic mice treated with control or anti-LAIR1 antagonist antibodies indicates that LAIR1 signaling blockade increased the numbers of CD4 memory T cells and inflammatory macrophages, but decreased those of pro-tumor macrophages, regulatory T cells, and plasmacytoid dendritic cells. Importantly, the LAIR1 blockade by the antagonistic antibody inhibited the activity of immunosuppressive myeloid cells and reactivated T cells from cancer patients in vitro and impeded tumor metastasis in a humanized mouse model. Blocking LAIR1 signaling in immune cells represents a promising strategy for development of anti-cancer immunotherapy.

Antibody therapeutics for epithelial ovarian cancer.

INTRODUCTION: High-grade serous ovarian carcinoma (HGSC) is an aggressive subtype of epithelial ovarian carcinoma (EOC) and remains the most lethal gynecologic cancer. A lack of effective and tolerable therapeutic options and nonspecific symptoms at presentation with advanced stage of disease are among the challenges in the management of the disease. AREAS COVERED: An overview of ovarian cancer, followed by a discussion of the current therapeutic regimes and challenges that arise during and after the treatment of EOC. We discuss different formats of antibody therapeutics and their usage in targeting validated targets implicated in ovarian cancer, as well as three emerging novel proteins as examples recently implicated in their contribution to adaptive resistance in ovarian cancer. EXPERT OPINION: Antibody therapeutics allow for a unique and effective way to target proteins implicated in cancer and other diseases, and have the potential to radically change the outcomes of patients suffering from ovarian cancer. The vast array of targets that have been implicated in ovarian cancer and yet the lack of effective therapeutic options for patients further stresses the importance of discovering novel proteins that can be targeted, as well as predictive biomarkers that can inform the stratification of patients into treatment-specific populations.

A tetravalent TREM2 agonistic antibody reduced amyloid pathology in a mouse model of Alzheimer's disease.

Triggering receptor expressed on myeloid cells 2 (TREM2) plays crucial roles in Alzheimer's disease (AD) by regulating microglia migration toward, and phagocytosis of oligomeric amyloid-ß (oAß) and amyloid plaques. Studies in rodent models of AD have shown that mice with increased TREM2 expression have reduced amyloid pathology. Here, we identified a TREM2 agonist monoclonal Ab (Ab18) by panning a phage-displayed single-chain variable fragment Ab library. By engineering the bivalent immunoglobulin G1 (IgG1) to tetra-variable domain immunoglobulin (TVD-Ig), we further increased the TREM2 activation by 100-fold. Stronger TREM2 activation led to enhanced microglia phagocytosis of the oAß-lipid complex, migration toward oAß, and improved microglia survival in vitro. Mechanistic studies showed increased TREM2 clustering on microglia by the tetravalent Ab18 TVD-Ig without altering microglial TREM2 amount. An engineered bispecific Ab targeting TREM2 and transferrin receptor (TfR; Ab18 TVD-Ig/αTfR) improved Ab brain entry by more than 10-fold with a broad brain parenchyma distribution. Weekly treatment of 5XFAD mice (a model of AD) with Ab18 TVD-Ig/αTfR showed a considerable reduction of amyloid burden with increased microglia migration to and phagocytosis of amyloid plaques, improved synaptic and neuronal marker intensity, improved cognitive functions, reduced endogenous tau hyperphosphorylation, and decreased phosphorylated neurofilament H immunostaining. This study demonstrated the feasibility of engineering multivalent TREM2 agonistic Ab coupled with TfR-mediated brain delivery to enhance microglia functions and reduce amyloid pathology in vitro and in vivo. This Ab engineering approach enables the development of effective TREM2-targeting therapies for AD.

Engineering SARS-CoV-2 specific cocktail antibodies into a bispecific format improves neutralizing potency and breadth.

One major limitation of neutralizing antibody-based COVID-19 therapy is the requirement of costly cocktails to reduce emergence of antibody resistance. Here we engineer two bispecific antibodies (bsAbs) using distinct designs and compared them with parental antibodies and their cocktail. Single molecules of both bsAbs block the two epitopes targeted by parental antibodies on the receptor-binding domain (RBD). However, bsAb with the IgG-(scFv)2 design (14-H-06) but not the CrossMAb design (14-crs-06) shows increased antigen-binding and virus-neutralizing activities against multiple SARS-CoV-2 variants as well as increased breadth of neutralizing activity compared to the cocktail. X-ray crystallography and cryo-EM reveal distinct binding models for individual cocktail antibodies, and computational simulations suggest higher inter-spike crosslinking potentials by 14-H-06 than 14-crs-06. In mouse models of infections by SARS-CoV-2 and multiple variants, 14-H-06 exhibits higher or equivalent therapeutic efficacy than the cocktail. Rationally engineered bsAbs represent a cost-effective alternative to antibody cocktails and a promising strategy to improve potency and breadth.

Impairment of IgG Fc functions promotes tumor progression and suppresses NK cell antitumor actions.

Natural killer (NK) cells mediate antibody dependent cytotoxic killing of cancer cells via cross-linking FcγR on NK cells with IgG-Fc. Studies have shown that the single-hinge cleaved IgGs (scIgGs) have dysfunctional Fc and failed engagement with FcγRs on immune cells. However, little is known about how scIgGs impact on antitumor immunity in the tumor microenvironment. In this study, we revealed a significant association of tumor scIgGs with tumor progression and poor outcomes of breast cancer patients (n = 547). Using multiple mouse tumor models, we demonstrated that tumor scIgGs reduced NK cell cytotoxic activities and resulted in aggressive tumor progression. We further showed that an anti-hinge specific monoclonal antibody (AHA) rescued the dysfunctional Fc in scIgGs by providing a functional Fc and restored NK cell cytotoxic activity. These findings point to a novel immunotherapeutic strategy to enhance Fc engagement with FcγRs for activation of anticancer immunity.

Discovery and engineering of an anti-TREM2 antibody to promote amyloid plaque clearance by microglia in 5XFAD mice.

Triggering receptor expressed on myeloid cells 2 (TREM2) plays a crucial role in regulating microglial functions and removal of amyloid plaques in Alzheimer's disease (AD). However, therapeutics based on this knowledge have not been developed due to the low antibody brain penetration and weak TREM2 activation. In this study, we engineered a TREM2 bispecific antibody to potently activate TREM2 and enter the brain. To boost TREM2 activation, we increased the valency of bivalent anti-TREM2 Ab2 IgG to tetra-variable domain immunoglobulin (TVD-Ig), thus improving the EC50 of amyloid-ß oligomer (oAß)-lipid microglial phagocytosis by more than 100-fold. Ab2 TVD-Ig treatment also augmented both microglia migration toward oAß and microglia survival by 100-fold over the bivalent IgG antibody. By targeting the transferrin receptor (TfR), the brain-penetrating Ab2 TVD-Ig/αTfR bispecific antibody realized broad brain parenchyma distribution with a 10-fold increase in brain antibody concentration. Ab2 TVD-Ig/αTfR treatment of 5-month-old 5XFAD mice significantly boosted microglia-plaque interactions and enhanced amyloid plaque phagocytosis by microglia. Thus, potent TREM2 activation by a multivalent agonist antibody coupled with TfR-mediated brain entry can boost microglia clearance of amyloid plaques, which suggests the antibody has potential as an AD treatment.List of abbreviations AD: Alzheimer's disease; Ab: antibody; APOE: apolipoprotein E; Aß: amyloid beta; BBB: blood-brain barrier; BLI: bio-layer interferometry; CNS: central nervous system; CSF: colony-stimulating factor; CytoD: cytochalasin d; DAM: microglia type associated with neurodegenerative diseases; DAP12: DNAX-activation protein 12; TVD-Ig: tetra-variable domain immunoglobulin; ECD: extracellular domain; ELISA: enzyme-linked immunoassay; ESC: embryonic stem cell; hMGLs: human embryonic stem cell-derived microglia-like lines; IBA1: ionized calcium-binding adaptor molecule 1; ITAM: immunoreceptor tyrosine-based activation motif; KiH: knob-into-hole; NFAT: nuclear factor of activated t-cells; PC: phosphatidylcholine; PK: pharmacokinetics; PS: phosphatidylserine; pSYK: phosphorylated spleen tyrosine kinase; scFv: single-chain variable fragment; SEC: size-exclusion chromatography; sTREM2: soluble triggering receptor expressed on myeloid cells 2; SYK: spleen tyrosine kinase; TfR: transferrin receptor; TREM2: triggering receptor expressed on myeloid cells 2.