Kappa-on-Heavy (KoH) bodies are a distinct class of fully-human antibody-like therapeutic agents with antigen-binding properties.

We describe a Kappa-on-Heavy (KoH) mouse that produces a class of highly diverse, fully human, antibody-like agents. This mouse was made by replacing the germline variable sequences of both the Ig heavy-chain (IgH) and Ig kappa (IgK) loci with the human IgK germline variable sequences, producing antibody-like molecules with an antigen binding site made up of 2 kappa variable domains. These molecules, named KoH bodies, structurally mimic naturally existing Bence-Jones light-chain dimers in their variable domains and remain wild-type in their antibody constant domains. Unlike artificially diversified, nonimmunoglobulin alternative scaffolds (e.g., DARPins), KoH bodies consist of a configuration of normal Ig scaffolds that undergo natural diversification in B cells. Monoclonal KoH bodies have properties similar to those of conventional antibodies but exhibit an enhanced ability to bind small molecules such as the endogenous cardiotonic steroid marinobufagenin (MBG) and nicotine. A comparison of crystal structures of MBG bound to a KoH Fab versus a conventional Fab showed that the KoH body has a much deeper binding pocket, allowing MBG to be held 4 Å further down into the combining site between the 2 variable domains.

Mechanisms of anaphylaxis in human low-affinity IgG receptor locus knock-in mice.

BACKGROUND: Anaphylaxis can proceed through distinct IgE- or IgG-dependent pathways, which have been investigated in various mouse models. We developed a novel mouse strain in which the human low-affinity IgG receptor locus, comprising both activating (hFcγRIIA, hFcγRIIIA, and hFcγRIIIB) and inhibitory (hFcγRIIB) hFcγR genes, has been inserted into the equivalent murine locus, corresponding to a locus swap. OBJECTIVE: We sought to determine the capabilities of hFcγRs to induce systemic anaphylaxis and identify the cell types and mediators involved. METHODS: hFcγR expression on mouse and human cells was compared to validate the model. Passive systemic anaphylaxis was induced by injection of heat-aggregated human intravenous immunoglobulin and active systemic anaphylaxis after immunization and challenge. Anaphylaxis severity was evaluated based on hypothermia and mortality. The contribution of receptors, mediators, or cell types was assessed based on receptor blockade or depletion. RESULTS: The human-to-mouse low-affinity FcγR locus swap engendered hFcγRIIA/IIB/IIIA/IIIB expression in mice comparable with that seen in human subjects. Knock-in mice were susceptible to passive and active anaphylaxis, accompanied by downregulation of both activating and inhibitory hFcγR expression on specific myeloid cells. The contribution of hFcγRIIA was predominant. Depletion of neutrophils protected against hypothermia and mortality. Basophils contributed to a lesser extent. Anaphylaxis was inhibited by platelet-activating factor receptor or histamine receptor 1 blockade. CONCLUSION: Low-affinity FcγR locus-switched mice represent an unprecedented model of cognate hFcγR expression. Importantly, IgG-related anaphylaxis proceeds within a native context of activating and inhibitory hFcγRs, indicating that, despite robust hFcγRIIB expression, activating signals can dominate to initiate a severe anaphylactic reaction.

Immuno-PET Monitoring of Lymphocytes Using the CD8-Specific Antibody REGN5054.

Assessment of immune-cell subsets within the tumor immune microenvironment is a powerful approach to better understand cancer immunotherapy responses. However, the use of biopsies to assess the tumor immune microenvironment poses challenges, including the potential for sampling error, restricted sampling over time, and inaccessibility of some tissues/organs, as well as the fact that single biopsy analyses do not reflect discordance across multiple intrapatient tumor lesions. Immuno-positron emission tomography (PET) presents a promising translational imaging approach to address the limitations and assess changes in the tumor microenvironment. We have developed 89Zr-DFO-REGN5054, a fully human CD8A-specific antibody conjugate, to assess CD8+ tumor-infiltrating lymphocytes (TIL) pre- and posttherapy. We used multiple assays, including in vitro T-cell activation, proliferation, and cytokine production, and in vivo viral clearance and CD8 receptor occupancy, to demonstrate that REGN5054 has minimal impact on T-cell activity. Preclinical immuno-PET studies demonstrated that 89Zr-DFO-REGN5054 specifically detected CD8+ T cells in lymphoid tissues of CD8-genetically humanized immunocompetent mice (VelociT mice) and discerned therapy-induced changes in CD8+ TILs in two models of response to a CD20xCD3 T-cell activating bispecific antibody (REGN1979, odronextamab). Toxicology studies in cynomolgus monkeys showed no overt toxicity, and immuno-PET imaging in cynomolgus monkeys demonstrated dose-dependent clearance and specific targeting to lymphoid tissues. This work supports the clinical investigation of 89Zr-DFO-REGN5054 to monitor T-cell responses in patients undergoing cancer immunotherapy.

Humanization of T cell-mediated immunity in mice.

Despite the enormous promise of T cell therapies, the isolation and study of human T cell receptors (TCRs) of dedicated specificity remains a major challenge. To overcome this limitation, we generated mice with a genetically humanized system of T cell immunity. We used VelociGene technology to replace the murine TCRαß variable regions, along with regions encoding the extracellular domains of co-receptors CD4 and CD8, and major histocompatibility complex (MHC) class I and II, with corresponding human sequences. The resulting "VelociT" mice have normal myeloid and lymphoid immune cell populations, including thymic and peripheral αß T cell subsets comparable with wild-type mice. VelociT mice expressed a diverse TCR repertoire, mounted functional T cell responses to lymphocytic choriomeningitis virus infection, and could develop experimental autoimmune encephalomyelitis. Immunization of VelociT mice with human tumor-associated peptide antigens generated robust, antigen-specific responses and led to identification of a TCR against tumor antigen New York esophageal squamous cell carcinoma-1 with potent antitumor activity. These studies demonstrate that VelociT mice mount clinically relevant T cell responses to both MHC-I­ and MHC-II­restricted antigens, providing a powerful new model for analyzing T cell function in human disease. Moreover, VelociT mice are a new platform for de novo discovery of therapeutic human TCRs.

An engineered human Fc domain that behaves like a pH-toggle switch for ultra-long circulation persistence.

The pharmacokinetic properties of antibodies are largely dictated by the pH-dependent binding of the IgG fragment crystallizable (Fc) domain to the human neonatal Fc receptor (hFcRn). Engineered Fc domains that confer a longer circulation half-life by virtue of more favorable pH-dependent binding to hFcRn are of great therapeutic interest. Here we developed a pH Toggle switch Fc variant containing the L309D/Q311H/N434S (DHS) substitutions, which exhibits markedly improved pharmacokinetics relative to both native IgG1 and widely used half-life extension variants, both in conventional hFcRn transgenic mice and in new knock-in mouse strains. engineered specifically to recapitulate all the key processes relevant to human antibody persistence in circulation, namely: (i) physiological expression of hFcRn, (ii) the impact of hFcγRs on antibody clearance and (iii) the role of competing endogenous IgG. DHS-IgG retains intact effector functions, which are important for the clearance of target pathogenic cells and also has favorable developability.

Publisher Correction: An engineered human Fc domain that behaves like a pH-toggle switch for ultra-long circulation persistence.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A novel bispecific antibody platform to direct complement activity for efficient lysis of target cells.

Harnessing complement-mediated cytotoxicity by therapeutic antibodies has been limited because of dependency on size and density of antigen, structural constraints resulting from orientation of antibody binding, and blockade of complement activation by inhibitors expressed on target cells. We developed a modular bispecific antibody platform that directs the complement-initiating protein C1q to target cells, increases local complement deposition and induces cytotoxicity against target antigens with a wide-range of expression. The broad utility of this approach to eliminate both prokaryotic and eukaryotic cells was demonstrated by pairing a unique C1q-recruiting arm with multiple targeting arms specific for Staphylococcus aureus, Pseudomonas aeruginosa, B-cells and T-cells, indicating applicability for diverse indications ranging from infectious diseases to cancer. Generation of C1q humanized mice allowed for demonstration of the efficacy of this approach to clear disease-inducing cells in vivo. In summary, we present a novel, broadly applicable, and versatile therapeutic modality for targeted cell depletion.

Demyelination, astrogliosis, and accumulation of ubiquitinated proteins, hallmarks of CNS disease in hsf1-deficient mice.

The heat shock transcription factors (Hsfs) are responsible for the heat shock response, an evolutionarily conserved process for clearance of damaged and aggregated proteins. In organisms such as Caenorhabditis elegans, which contain a single Hsf, reduction in the level of Hsf is associated with the appearance of age-related phenotypes and increased accumulation of protein aggregates. Mammalian cells express three hsfs (hsf1, hsf2, hsf4) and their role in CNS homeostasis remains unclear. In this study, we examined the effects of deletion of single or multiple hsf genes in the CNS using mutant mice. Our results show that hsf1-/- mice display progressive myelin loss that accompanies severe astrogliosis and this is exacerbated in the absence of either the hsf2 or hsf4 gene. Magnetic resonance imaging and behavioral studies indicate reduction in the white matter tracts of the corpus callosum, and deficiencies in motor activity, respectively, in aged hsf1-/- mice. Concomitantly, hsf1-/- aged CNS exhibit increased activated microglia and apoptotic cells that are mainly positive for GFAP, an astrocyte-specific marker. Studies based on the expression of short-lived ubiquitinated green fluorescent protein (GFPu) in living hsf1-/- cells indicate that they exhibit reduced ability to degrade ubiquitinated proteins, accumulate short-lived GFPu, and accumulate aggregates of the Huntington's model of GFP containing trinucleotide repeats (Q103-GFP). Likewise, hsf1-/- brain and astrocytes exhibit higher than wild-type levels of ubiquitinated proteins, increased levels of protein oxidation, and increased sensitivity to oxidative stress. These studies indicate a critical role for mammalian hsf genes, but specifically hsf1, in the quality control mechanisms and maintenance of CNS homeostasis during the organism's lifetime.

Heat shock transcription factor (Hsf)-4b recruits Brg1 during the G1 phase of the cell cycle and regulates the expression of heat shock proteins.

Human brahma-related gene 1(Brg1) is a subunit of the switching/sucrose non-fermenting (SWI/SNF) chromatin-remodeling complex and regulates transcription during cell growth and differentiation and has been found to be mutated in many types of human cancers. Mammalian heat shock factor 1 (Hsf1), which binds conserved sequences on the promoter of the hsp70 gene when cells are exposed to various stress stimuli, utilizes Brg1-SWI/SNF complexes and stimulates transcription in vitro at the level of initiation and elongation. In contrast to the stress-inducibility of Hsf1, in vitro transcribed/translated Hsf4b binds to the heat shock element (HSE) constitutively and loses its ability to bind HSEs following stress. The regulation of Hsf4b transcriptional activity in vivo remains unclear. Here, we present evidence that Hsf4b recruits Brg1 complexes to the promoters of heat shock proteins (HSPs) under physiological growth conditions. Furthermore, in an asynchronous cell population, the association of Hsf4b with Brg1 complexes is regulated in response to activation/inactivation of the extracellular signal regulated protein kinase 1/2 (ERK1/2) signaling pathway. Since Brg1 is also the target of mitogen-activated protein (MAP) kinases and other protein kinases and it is hyperphosphorylated and inactivated during the G2/M phase of the cell cycle, we tested whether the association of Hsf4b with Brg1 complexes is altered during the cell cycle. The results indicate that association of Hsf4b with Brg1 complexes is undetectable during G2/M; however, an Hsf4b interaction with Brg1 complexes is evident at 1-3 h after progression of cells into G1, where chromatin structure is presumed to be more accessible to transcriptional regulatory proteins. At this time, Hsf4b exhibits increased DNA-binding activity and is detectable on promoters of multiple Hsps. To determine the unique role of Hsf4b in stimulating the expression of Hsps during the cell cycle, experiments were conducted with mouse embryo fibroblasts (MEFs) deficient in individual Hsfs. The results indicate that in the absence of Hsf1 and Hsf2, Hsf4b expression in cells leads to increased ability of Hsf4b to bind HSE during G1, leading to enhanced synthesis of inducible Hsp70.

Critical role of Brg1 member of the SWI/SNF chromatin remodeling complex during neurogenesis and neural crest induction in zebrafish.

Brg1 is a member of the SWI/SNF chromatin-remodeling complex, and in some organisms Brg1 has been shown to interact with beta-catenin and positively control the TCF/LEF transcription factor that is located downstream of the Wnt signal transduction pathway. During development, TCF/LEF activity is critical during neurogenesis and head induction. In zebrafish, Brg1-deficient embryos exhibit retinal cell differentiation and eye defects; however, the role of Brg1 in neurogenesis and neural crest cell induction remains elusive. We used zebrafish deficient in Brg1 (yng) or Brg1 specific-morpholino oligonucleotide-mediated knockdown to analyze the embryonic requirements of Brg1. Our results indicate that reduction in Brg1 expression leads to the expansion of the forebrain-specific transcription factor, six3, and marked reduction in expression of the mid/hind-brain boundary and hind-brain genes, engrailed2 and krox20, respectively. At 12 hpf, the expression of neural crest specifiers are severely affected in Brg1-morpholino-injected embryos. These results suggest that Brg1 is involved in neural crest induction, which is critical for the development of neurons, glia, pigment cells, and craniofacial structures. Brg1 is a maternal factor, and brg1-deficient embryos bearing the yng mutation derived from heterozygote intercrosses exhibit lesser effects on neural crest-specific gene expression, but show defects in neurogenesis and neural crest cell differentiation. This is exhibited by the aberrant brain patterning, a reduction in the sensory neurons, and craniofacial defects. These results further elucidate the critical role for Brg1 in neurogenesis, neural crest induction, and differentiation.

Essential requirement for both hsf1 and hsf2 transcriptional activity in spermatogenesis and male fertility.

Heat shock factors (Hsfs) are major transactivators of heat shock proteins but are also involved in regulation of other genes active in embryonic development. High expression levels of Hsfs in mouse testis during development suggest a role for these factors in spermatogenesis, a cyclic process of spermatogonia cell-differentiation into mature spermatozoa. In contrast to hsf1(-/-) mice, which exhibit normal spermatogenesis, targeted disruption of hsf2 results in reduced testicular size but only a small impairment in male fertility. We show here that disruption of both hsf1 and hsf2 results in a more severe phenotype associated with male sterility due to severe defects in spermatogenesis. Earliest defects observed are the reduced number of germ cells in juvenile mice and germ cells that enter the meiotic prophase fail to progress beyond the pachytene stage. This was associated with a reduction or absence of transcription of genes critically involved in spermatogenesis. The findings suggest that additive or synergistic transcriptional activity of both hsf1 and hsf2 is required for normal mammalian spermatogenesis and male fertility.