The establishment of B cell-deficient Igh-J KO mouse model by gene editing and efficacy evaluation.

Over the last few years, immunotherapy has made significant progress in treating various cancers with therapeutic antibodies. However, therapeutic antibodies have been validated for inducing an unintended immune response in human and animal models, which leads to the emergence of anti-drug antibodies (ADAs) and affects their effectiveness and safety. In preclinical research, ADAs production by B cells may accelerate antibody metabolism and result in missing potential candidate molecules. Thus, it is urgent to develop preclinical models that remove only B cells without affecting the function of T and NK cells. Rearrangement of immunoglobulin heavy chain J gene fragment (Igh-J) is the first link in B cell development, and immunotherapies are currently leaning toward combination treatments with PD-1/PD-L1 antibodies, here we created humanized PD-1, PD-L1 and Igh-J knockout (hPD-1/hPD-L1, Igh-J KO) mice and validated by using the reported high immunogenicity drug M7824 (a protein designed to simultaneously block PD-L1 and TGF-ß pathways, poorly anti-tumor efficacy in immunocompetent mice). Phenotypic analysis revealed that human PD-1 and PD-L1 were detectable in hPD-1/hPD-L1, Igh-J KO mice, but not mouse IgM and IgD. Igh-J KO depleted B cells while increased the percentage of other immune cell types. Meanwhile, the humanization of PD-1/PD-L1 and Igh-J KO had neither effect on the overall development, differentiation, or distribution of T cell subtypes, nor on the activation of NK and T cells, indicating that mice can be used for T and NK-related immunotherapies. Furthermore, M7824 treatment of these B cell-deficient mice inhibited tumor growth significantly, with higher M7824 analog concentrations and lower ADA-positive rates. These findings demonstrate that Igh-J KO mice are an effective and stable preclinical model for testing drugs based on T and NK cells with high immunogenicity in vivo.

Strategies to mitigate the on- and off-target toxicities of recombinant immunotoxins: an antibody engineering perspective.

Targeted cancer therapies using immunotoxins have achieved remarkable efficacy in hematological malignancies. However, the clinical development of immunotoxins is also faced with many challenges like anti-drug antibodies and dose-limiting toxicity issues. Such a poor efficacy or safety ratio is also the major hurdle in the research and development of antibody-drug conjugates. From an antibody engineering perspective, various strategies were summarized or proposed to tackle the notorious on-target off-tumor toxicity issues, including passive strategy (XTENylation of immunotoxins) and active strategies (modulating the affinity and valency of the targeting moiety of immunotoxins, conditionally activating immunotoxins in the tumor microenvironments and reconstituting split toxin to reduce systemic toxicity, etc.). By modulating the functional characteristics of the targeting moiety and the toxic moiety of immunotoxins, selective tumor targeting can be augmented while sparing the healthy cells in normal tissues expressing the same target of interest. If successful, the improved therapeutic index will likely help to address the dose-limiting toxicities commonly observed in the clinical trials of various immunotoxins.

A high-throughput single cell-based antibody discovery approach against the full-length SARS-CoV-2 spike protein suggests a lack of neutralizing antibodies targeting the highly conserved S2 domain.

Coronavirus disease 2019 pandemic continues globally with a growing number of infections, but there are currently no effective antibody drugs against the virus. In addition, 90% amino acid sequence identity between the S2 subunit of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-CoV S proteins attracts us to examine S2-targeted cross-neutralizing antibodies that are not yet well defined. We therefore immunized RenMab mice with the full-length S protein and constructed a high-throughput antibody discovery method based on single-cell sequencing technology to isolate SARS-CoV-2 S-targeted neutralizing antibodies and cross-neutralizing antibodies against the S2 region of SARS-CoV-2/SARS-CoV S. Diversity of antibody sequences in RenMab mice and consistency in B-cell immune responses between RenMab mice and humans enabled screening of fully human virus-neutralizing antibodies. From all the frequency >1 paired clonotypes obtained from single-cell V(D)J sequencing, 215 antibodies with binding affinities were identified and primarily bound S2. However, only two receptor-binding domain-targeted clonotypes had neutralizing activity against SARS-CoV-2. Moreover, 5' single-cell RNA sequencing indicated that these sorted splenic B cells are mainly plasmablasts, germinal center (GC)-dependent memory B-cells and GC B-cells. Among them, plasmablasts and GC-dependent memory B-cells were considered the most significant possibility of producing virus-specific antibodies. Altogether, using a high-throughput single cell-based antibody discovery approach, our study highlighted the challenges of developing S2-binding neutralizing antibodies against SARS-CoV-2 and provided a novel direction for the enrichment of antigen-specific B-cells.

Generation of a uniform thymic malignant lymphoma model with C57BL/6J p53 gene deficient mice.

Lymphoma is the third most common cancer diagnosed in children, and T-cell lymphoma has the worst prognosis based on clinical observations. To date, a lymphoma model with uniform penetrance has not yet been developed. In this study, we generated a p53 deficient mouse model by targeting embryonic stem cells derived from a C57BL/6J mouse strain. Homozygous p53 deficient mice exhibited a higher rate of spontaneous tumorigenesis, with a high spontaneous occurrence rate (93.3%) of malignant lymphoma. Because tumor models with high phenotypic consistency are currently needed, we generated a lymphoma model by a single intraperitoneal injection of 37.5 or 75 mg/kg N-methyl-N-nitrosourea to p53 deficient mice. Lymphoma and retinal degeneration occurred in 100% of p53 +/- mice administered with higher concentrations of N-methyl-N-nitrosourea, a much greater response than those of previously reported models. The main anatomic sites of lymphoma were the thymus, spleen, bone marrow, and lymph nodes. Both induced and spontaneous lymphomas in the thymus and spleen stained positive for CD3 antigen, and flow cytometry detected positive CD4 and/or CD8 cells. Based on our observations and previous data, we hypothesize that mice with a B6 background are prone to lymphomagenesis.

Antibody heavy chain CDR3 length-dependent usage of human IGHJ4 and IGHJ6 germline genes.

Therapeutic antibody discovery using synthetic diversity has been proved productive, especially for target proteins not suitable for traditional animal immunization-based antibody discovery approaches. Recently, many lines of evidences suggest that the quality of synthetic diversity design limits the development success of synthetic antibody hits. The aim of our study is to understand the quality limitation and to properly address the challenges with a better design. Using VH3-23 as a model framework, we observed and quantitatively mapped CDR-H3 loop length-dependent usage of human IGHJ4 and IGHJ6 germline genes in the natural human immune repertoire. Skewed usage of DH2-JH6 and DH3-JH6 rearrangements was quantitatively determined in a CDR-H3 length-dependent manner in natural human antibodies with long CDR-H3 loops. Structural modeling suggests choices of JH help to stabilize antibody CDR-H3 loop and JH only partially contributes to the paratope. Our observations shed light on the design of next-generation synthetic diversity with improved probability of success.

Three epitope-distinct human antibodies from RenMab mice neutralize SARS-CoV-2 and cooperatively minimize the escape of mutants.

Coronavirus disease 2019 (COVID-19), a pandemic disease caused by the newly emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused more than 3.8 million deaths to date. Neutralizing antibodies are effective therapeutic measures. However, many naturally occurring mutations at the receptor-binding domain (RBD) have emerged, and some of them can evade existing neutralizing antibodies. Here, we utilized RenMab, a novel mouse carrying the entire human antibody variable region, for neutralizing antibody discovery. We obtained several potent RBD-blocking antibodies and categorized them into four distinct groups by epitope mapping. We determined the involved residues of the epitope of three representative antibodies by cryo-electron microscopy (Cryo-EM) studies. Moreover, we performed neutralizing experiments with 50 variant strains with single or combined mutations and found that the mixing of three epitope-distinct antibodies almost eliminated the mutant escape. Our study provides a sound basis for the rational design of fully human antibody cocktails against SARS-CoV-2 and pre-emergent coronaviral threats.

Functional comparison of SARS-CoV-2 with closely related pangolin and bat coronaviruses.

The origin and intermediate host for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is yet to be determined. Coronaviruses found to be closely related to SARS-CoV-2 include RaTG13 derived from bat and two clusters (PCoV-GD and PCoV-GX) of coronaviruses identified in pangolin. Here, we studied the infectivity and antigenicity patterns of SARS-CoV-2 and the three related coronaviruses. Compared with the other three viruses, RaTG13 showed almost no infectivity to a variety of cell lines. The two pangolin coronaviruses and SARS-CoV-2 showed similar infectious activity. However, in SARS-CoV-2-susceptible cell lines, the pangolin coronaviruses presented even higher infectivity. The striking difference between the SARS-CoV-2 and pangolin coronaviruses is that the latter can infect porcine cells, which could be partially attributed to an amino acid difference at the position of 498 of the spike protein. The infection by SARS-CoV-2 was mainly mediated by Furin and TMPRSS2, while PCoV-GD and PCoV-GX mainly depend on Cathepsin L. Extensive cross-neutralization was found between SARS-CoV-2 and PCoV-GD. However, almost no cross-neutralization was observed between PCoV-GX and SARS-CoV-2 or PCoV-GD. More attention should be paid to pangolin coronaviruses and to investigate the possibility of these coronaviruses spreading across species to become zoonoses among pigs or humans.

SARS-CoV-2 501Y.V2 variants lack higher infectivity but do have immune escape.

The 501Y.V2 variants of SARS-CoV-2 containing multiple mutations in spike are now dominant in South Africa and are rapidly spreading to other countries. Here, experiments with 18 pseudotyped viruses showed that the 501Y.V2 variants do not confer increased infectivity in multiple cell types except for murine ACE2-overexpressing cells, where a substantial increase in infectivity was observed. Notably, the susceptibility of the 501Y.V2 variants to 12 of 17 neutralizing monoclonal antibodies was substantially diminished, and the neutralization ability of the sera from convalescent patients and immunized mice was also reduced for these variants. The neutralization resistance was mainly caused by E484K and N501Y mutations in the receptor-binding domain of spike. The enhanced infectivity in murine ACE2-overexpressing cells suggests the possibility of spillover of the 501Y.V2 variants to mice. Moreover, the neutralization resistance we detected for the 501Y.V2 variants suggests the potential for compromised efficacy of monoclonal antibodies and vaccines.

A Mouse Model of SARS-CoV-2 Infection and Pathogenesis.

Since December 2019, a novel coronavirus SARS-CoV-2 has emerged and rapidly spread throughout the world, resulting in a global public health emergency. The lack of vaccine and antivirals has brought an urgent need for an animal model. Human angiotensin-converting enzyme II (ACE2) has been identified as a functional receptor for SARS-CoV-2. In this study, we generated a mouse model expressing human ACE2 (hACE2) by using CRISPR/Cas9 knockin technology. In comparison with wild-type C57BL/6 mice, both young and aged hACE2 mice sustained high viral loads in lung, trachea, and brain upon intranasal infection. Although fatalities were not observed, interstitial pneumonia and elevated cytokines were seen in SARS-CoV-2 infected-aged hACE2 mice. Interestingly, intragastric inoculation of SARS-CoV-2 was seen to cause productive infection and lead to pulmonary pathological changes in hACE2 mice. Overall, this animal model described here provides a useful tool for studying SARS-CoV-2 transmission and pathogenesis and evaluating COVID-19 vaccines and therapeutics.

Rosa26-targeted sheep gene knock-in via CRISPR-Cas9 system.

Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Technologies that facilitate specific and precise genome editing, such as knock-in, are critical for determining the functions of genes and for understanding fundamental biological processes. The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in mammals. Rosa26 gene can encode a non-essential nuclear RNA in almost all organizations, and become a hot point of exogenous gene insertion. Here, we describe efficient, precise CRISPR/Cas9-mediated Integration using a donor vector with tGFP sequence targeted in the sheep genomic Rosa26 locus. We succeeded in integrating with high efficiency an exogenous tGFP (turboGFP) gene into targeted genes in frame. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic sheep.

Bioluminescent imaging of vaccinia virus infection in immunocompetent and immunodeficient rats as a model for human smallpox.

Due to the increasing concern of using smallpox virus as biological weapons for terrorist attack, there is renewed interest in studying the pathogenesis of human smallpox and development of new therapies. Animal models are highly demanded for efficacy and safety examination of new vaccines and therapeutic drugs. Here, we demonstrated that both wild type and immunodeficient rats infected with an engineered vaccinia virus carrying Firefly luciferase reporter gene (rTV-Fluc) could recapitulate infectious and clinical features of human smallpox. Vaccinia viral infection in wild type Sprague-Dawley (SD) rats displayed a diffusible pattern in various organs, including liver, head and limbs. The intensity of bioluminescence generated from rTV-Fluc correlated well with viral loads in tissues. Moreover, neutralizing antibodies had a protective effect against virus reinfection. The recombination activating gene 2 (Rag2) knockout rats generated by transcription activator-like effector nucleases (TALENs) technology were further used to examine the infectivity of the rTV-Fluc in immunodeficient populations. Here we demonstrated that Rag2-/- rats were more susceptible to rTV-Fluc than SD rats with a slower virus clearance rate. Therefore, the rTV-Fluc/SD rats and rTV-Fluc/Rag2-/- rats are suitable visualization models, which recapitulate wild type or immunodeficient populations respectively, for testing human smallpox vaccine and antiviral drugs.

Visualization of mucosal homeostasis via single- and multiphoton intravital fluorescence microscopy.

FIVM has provided many insights into the regulation of immunity. We report the validation of an approach for visualizing murine small bowel via single- and multiphoton FIVM. Tissue damage is limited to ∼200 µm, immediately adjacent to the incision, as confirmed by intravital PI staining. Treatment with 10 KDa dextran-FITC and 70 KDa dextran-TR confirms that perfusion is intact. Selective filtration of 10 KDa but not 70 KDa dextran from the blood indicated that kidney function is also intact. Interestingly, lamina propria vasculature is semipermeable to 10 KDa dextran. Next, reporter mice expressing egfp from the CX3CR1 locus, egfp from the FoxP3 locus, or RFP from the IL-17F locus were used to track DC subsets, FoxP3(+) Tregs, or Th17f cells, respectively. Resident cx3cr1(+/egfp) cells were sessile but actively probed the surrounding microenvironment. Both T cell populations patrol the lamina propria, but the Th17f cells migrate more rapidly than Tregs. Together, these data demonstrate intact vascular perfusion, while intravitally visualizing the mucosal surface of the small bowel. Lastly, the cx3cr1(+) DCs and T cells display activity similar to that found in steady-state, secondary lymphoid organs.

CXCR4 acts as a costimulator during thymic beta-selection.

Passage through the beta-selection developmental checkpoint requires productive rearrangement of segments of the T cell antigen receptor-beta gene (Tcrb) and formation of a pre-TCR on the surface of CD4(-)CD8(-) thymocytes. How other receptors influence betabeta-selection is less well understood. Here we define a new role for the chemokine receptor CXCR4 during T cell development. CXCR4 functionally associated with the pre-TCR and influenced beta-selection by regulating the steady-state localization of immature thymocytes in thymic subregions, by facilitating optimal pre-TCR-induced survival signals, and by promoting thymocyte proliferation. We also characterize functionally relevant signaling molecules downstream of CXCR4 and the pre-TCR in thymocytes. Our data designate CXCR4 as a costimulator of the pre-TCR during beta-selection.

How punctual ablation of regulatory T cells unleashes an autoimmune lesion within the pancreatic islets.

CD4(+)Foxp3(+) regulatory T cells (Treg cells) are known to control the progression of autoimmune diabetes, but when, where, and how they exert their influence in this context are questions still under vigorous debate. Exploiting a transgene encoding the human diphtheria toxin receptor, we punctually and specifically ablated Foxp3(+) cells in the BCD2.5/NOD mouse model of autoimmune diabetes. Strikingly, overt disease developed within 3 days. The earliest detectable event was the activation of natural killer (NK) cells directly within the insulitic lesion, particularly the induction of Ifng gene expression within 7 hours of Treg cell ablation. Interferon-gamma had a strong impact on the gene-expression program of the local CD4(+) T effector cell population, unleashing it to aggressively attack the islets, which was required for the development of diabetes. Thus, Treg cells regulate pancreatic autoimmunity in situ through control of a central innate immune system player, NK cells.

Limited tumor infiltration by activated T effector cells restricts the therapeutic activity of regulatory T cell depletion against established melanoma.

Interference with inhibitory immunological checkpoints controlling T cell activation provides new opportunities to augment cancer immunotherapies. Whereas cytotoxic T lymphocyte-associated antigen-4 blockade has shown promising preclinical and clinical results, therapeutic CD4(+)CD25(+) T reg cell depletion has failed to consistently enhance immune-based therapies. Using B16/BL6, a transplantable murine melanoma model, we show a dichotomy between the effects of T reg cell depletion on tumor rejection dependent on whether depletion occurs before (prophylactic) or after (therapeutic) tumor engraftment. Failure to promote rejection with therapeutic depletion is not related to lack of T reg cell depletion, to elimination of CD25(+) effector T cells, or to a failure to enhance systemic antitumor T cell responses, but correlates with failure of effector cells to infiltrate the tumor and increase the intratumor ratio of effector T cell/T reg cell. Finally, systemic antitumor responses generated upon therapeutic T reg cell depletion are significantly stronger than those generated in the presence of T reg cells, and are capable of eliciting rejection of established tumors after transfer into immunoablated recipients receiving combination immunotherapy. The data demonstrate a dissociation between measurable systemic responses and tumor rejection during CD25-directed T reg cell depletion, and suggest an alternative, clinically applicable strategy for the treatment of established tumors.

TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function.

T helper cells that produce IL-17 (T(H)17 cells) promote autoimmunity in mice and have been implicated in the pathogenesis of human inflammatory diseases. At mucosal surfaces, T(H)17 cells are thought to protect the host from infection, whereas regulatory T (T(reg)) cells control immune responses and inflammation triggered by the resident microflora. Differentiation of both cell types requires transforming growth factor-beta (TGF-beta), but depends on distinct transcription factors: RORgammat (encoded by Rorc(gammat)) for T(H)17 cells and Foxp3 for T(reg) cells. How TGF-beta regulates the differentiation of T cells with opposing activities has been perplexing. Here we demonstrate that, together with pro-inflammatory cytokines, TGF-beta orchestrates T(H)17 cell differentiation in a concentration-dependent manner. At low concentrations, TGF-beta synergizes with interleukin (IL)-6 and IL-21 (refs 9-11) to promote IL-23 receptor (Il23r) expression, favouring T(H)17 cell differentiation. High concentrations of TGF-beta repress IL23r expression and favour Foxp3+ T(reg) cells. RORgammat and Foxp3 are co-expressed in naive CD4+ T cells exposed to TGF-beta and in a subset of T cells in the small intestinal lamina propria of the mouse. In vitro, TGF-beta-induced Foxp3 inhibits RORgammat function, at least in part through their interaction. Accordingly, lamina propria T cells that co-express both transcription factors produce less IL-17 (also known as IL-17a) than those that express RORgammat alone. IL-6, IL-21 and IL-23 relieve Foxp3-mediated inhibition of RORgammat, thereby promoting T(H)17 cell differentiation. Therefore, the decision of antigen-stimulated cells to differentiate into either T(H)17 or T(reg) cells depends on the cytokine-regulated balance of RORgammat and Foxp3.

The cytosolic endopeptidase, thimet oligopeptidase, destroys antigenic peptides and limits the extent of MHC class I antigen presentation.

Most antigenic peptides presented on MHC class I molecules are generated by proteasomes during protein breakdown. It is unknown whether these peptides are protected from destruction by cytosolic peptidases. In cytosolic extracts, most antigenic peptides are degraded by the metalloendopeptidase, thimet oligopeptidase (TOP). We therefore examined whether TOP destroys antigenic peptides in vivo. When TOP was overexpressed in cells, class I presentation of antigenic peptides was reduced. In contrast, TOP overexpression didn't reduce presentation of peptides generated in the endoplasmic reticulum or endosomes. Conversely, preventing TOP expression with siRNA enhanced presentation of antigenic peptides. TOP therefore plays an important role in vivo in degrading peptides released by proteasomes and is a significant factor limiting the extent of antigen presentation.