A CD22-Shp1 phosphatase axis controls integrin ß7 display and B cell function in mucosal immunity.

The integrin α4ß7 selectively regulates lymphocyte trafficking and adhesion in the gut and gut-associated lymphoid tissue (GALT). Here, we describe unexpected involvement of the tyrosine phosphatase Shp1 and the B cell lectin CD22 (Siglec-2) in the regulation of α4ß7 surface expression and gut immunity. Shp1 selectively inhibited ß7 endocytosis, enhancing surface α4ß7 display and lymphocyte homing to GALT. In B cells, CD22 associated in a sialic acid-dependent manner with integrin ß7 on the cell surface to target intracellular Shp1 to ß7. Shp1 restrained plasma membrane ß7 phosphorylation and inhibited ß7 endocytosis without affecting ß1 integrin. B cells with reduced Shp1 activity, lacking CD22 or expressing CD22 with mutated Shp1-binding or carbohydrate-binding domains displayed parallel reductions in surface α4ß7 and in homing to GALT. Consistent with the specialized role of α4ß7 in intestinal immunity, CD22 deficiency selectively inhibited intestinal antibody and pathogen responses.

CD22 blockade restores homeostatic microglial phagocytosis in ageing brains.

Microglia maintain homeostasis in the central nervous system through phagocytic clearance of protein aggregates and cellular debris. This function deteriorates during ageing and neurodegenerative disease, concomitant with cognitive decline. However, the mechanisms of impaired microglial homeostatic function and the cognitive effects of restoring this function remain unknown. We combined CRISPR-Cas9 knockout screens with RNA sequencing analysis to discover age-related genetic modifiers of microglial phagocytosis. These screens identified CD22, a canonical B cell receptor, as a negative regulator of phagocytosis that is upregulated on aged microglia. CD22 mediates the anti-phagocytic effect of α2,6-linked sialic acid, and inhibition of CD22 promotes the clearance of myelin debris, amyloid-ß oligomers and α-synuclein fibrils in vivo. Long-term central nervous system delivery of an antibody that blocks CD22 function reprograms microglia towards a homeostatic transcriptional state and improves cognitive function in aged mice. These findings elucidate a mechanism of age-related microglial impairment and a strategy to restore homeostasis in the ageing brain.

Deciphering the importance of culture pH on CD22 CAR T-cells characteristics.

BACKGROUND: Chimeric antigen receptor (CAR) T-cells have demonstrated significant efficacy in targeting hematological malignancies, and their use continues to expand. Despite substantial efforts spent on the optimization of protocols for CAR T-cell manufacturing, critical parameters of cell culture such as pH or oxygenation are rarely actively monitored during cGMP CAR T-cell generation. A comprehensive understanding of the role that these factors play in manufacturing may help in optimizing patient-specific CAR T-cell therapy with maximum benefits and minimal toxicity. METHODS: This retrospective study examined cell culture supernatants from the manufacture of CAR T-cells for 20 patients with B-cell malignancies enrolled in a phase 1/2 clinical trial of anti-CD22 CAR T-cells. MetaFLEX was used to measure supernatant pH, oxygenation, and metabolites, and a Bio-Plex assay was used to assess protein levels. Correlations were assessed between the pH of cell culture media throughout manufacturing and cell proliferation as well as clinical outcomes. Next-generation sequencing was conducted to examine gene expression profiles of the final CAR T-cell products. RESULTS: A pH level at the lower range of normal at the beginning of the manufacturing process significantly correlated with measures of T-cell expansion and metabolism. Stable or rising pH during the manufacturing process was associated with clinical response, whereas a drop in pH was associated with non-response. CONCLUSIONS: pH has potential to serve as an informative factor in predicting CAR T-cell quality and clinical outcomes. Thus, its active monitoring during manufacturing may ensure a more effective CAR T-cell product.

Siglec cis-ligands and their roles in the immune system.

Sialic acid-binding immunoglobulin-like lectins are a family of membrane molecules primarily expressed in immune cells. Most of them are inhibitory receptors containing immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic tail. On the cell surface, sialic acid-binding immunoglobulin-like lectins are mostly bound by sialylated glycans on membrane molecules expressed in the same cell (cis-ligands). Although ligands of sialic acid-binding immunoglobulin-like lectins are not efficiently identified by conventional methods such as immunoprecipitation, in situ labeling including proximity labeling is useful in identifying both cis-ligands and the sialylated ligands expressed by other cells (trans-ligands) of sialic acid-binding immunoglobulin-like lectins. Interaction of the inhibitory sialic acid-binding immunoglobulin-like lectins with cis-ligands including both those with and without signaling function modulates the inhibitory activity of sialic acid-binding immunoglobulin-like lectins by multiple different ways. This interaction also modulates signaling function of the cis-ligands. So far, little is known about the role of the interaction between sialic acid-binding immunoglobulin-like lectins and the cis-ligands. Nonetheless, recent studies showed that the inhibitory activity of CD22 (also known as Siglec-2) is regulated by endogenous ligands, most likely cis-ligands, differentially in resting B cells and those in which B-cell antigen receptor is ligated. This differential regulation plays a role in quality control of signaling-competent B cells and also partial restoration of B-cell antigen receptor signaling in immunodeficient B cells.

CD22 is a potential target of CAR-NK cell therapy for esophageal squamous cell carcinoma.

BACKGROUND: Chimeric antigen receptor NK (CAR-NK) cell therapy is one of the most promising immunotherapies. Although it has shown a significant therapeutic effect in hematologic malignancies, few successes have been obtained in solid tumors including esophageal squamous cell carcinoma (ESCC). The major reasons are lack of specific cell surface antigens and complex tumor microenvironment. Here we identify CD22, a well-known tumor surface marker in hematologic malignancies, is expressed in ESCC, possibly serving as a potential target of CAR-NK cell therapy. METHODS: The expression of 13 tumor cell surface antigens used clinically was analyzed in patients from The Cancer Genome Atlas (TCGA) database. Also, mRNA expression were detected in 2 ESCC cell lines and 2 patients samples by qCPR. Then according to Venn diagram, CD22 was selected for further investigation. Following this, the expression of CD22 by immunofluorescence (IF) in ESCC cell lines and by immunohistochemistry (IHC) in 87 cases of human ESCC samples was detected respectively. On the basis of H-score results, the correlation between CD22 expression and clinical parameters was analyzed. As a proof, the efficacy of CD22-targeted CAR-NK cells against ESCC cell lines was performed by a real-time cell analyzer (RTCA) platform. RESULTS: KYSE-140 and KYSE-150 cell lines displayed surface expression of CD22. IHC showed an 80.46% (70/87) positive rate in ESCC patient samples. Among these, cell membranous expression of CD22 was observed in 27.59% (24/87) patient samples. Through chi-square test, expression of CD22 in ESCC was associated with lymph node metastasis while it was no related to the depth of tumor invasion and clinical stage. Engineered CD22-targeted CAR-NK cells exhibited inhibitory growth capability against ESCC cell lines (p < 0.0001). CONCLUSIONS: CD22 is a potential tumor surface antigen capable of being targeted by CAR-NK cells in ESCC. And potential therapeutics for ESCC may be developed based on immune cells expressing anti-CD22 CAR. The study also indicates that CD22 CAR-NK cells could be used in other cancers and more in vivo experiments are needed.

Improved synthesis of CD22-binding sialosides and its application for further development of potent CD22 inhibitors.

CD22, one of the sialic acid-binding immunoglobulin-like lectins (Siglecs), regulates B lymphocyte signaling via its interaction with glycan ligands bearing the sequence Neu5Ac/Gcα(2→6)Gal. We have developed the synthetic sialoside GSC-718 as a ligand mimic for CD22 and identified it as a potent CD22 inhibitor. Although the synthesis of CD22-binding sialosides including GSC-718 has been reported by our group, the synthetic route was unfortunately not suitable for large-scale synthesis. In this study, we developed an improved scalable synthetic procedure for sialosides which utilized 1,5-lactam formation as a key step. The improved procedure yielded sialosides incorporating a series of aglycones at the C2 position. Several derivatives with substituted benzyl residues as aglycones were found to bind to mouse CD22 with affinity comparable to that of GSC-718. The new procedure developed in this study affords sialosides in sufficient quantities for cell-based assays, and will facilitate the search for promising CD22 inhibitors that have therapeutic potential.

CD22 blockade modulates microglia activity to suppress neuroinflammation following intracerebral hemorrhage.

Microglia are first responders to acute brain insults and initiate neuroinflammation to drive secondary tissue injury. Yet the key molecular switches in control of the inflammatory activity of microglia remain poorly understood. Intracerebral hemorrhage (ICH) is a devastating stroke subtype whereby a hematoma is formed within the brain parenchyma and associated with high mortality. Using a mouse model of ICH, we found upregulation of CD22 that predominantly occurred in microglia. Antibody blockade of CD22 led to a reduction in neurological deficits, brain lesion and hematoma volume. This was accompanied by reduced inflammatory activity, increased expression of alternative activation markers (CD206 and IL-10) and enhanced phagocytosis activity in microglia after ICH. CD22 blockade also led to an increase of phosphorylated SYK and AKT after ICH. Notably, the benefits of CD22 blockade were ablated in ICH mice subjected to microglial depletion with a colony-stimulating factor 1 receptor inhibitor PLX5622. Additionally, the protective effects of CD22 blockade was diminished in ICH mice receiving a SYK inhibitor R406. Together, our findings highlight CD22 as a key molecular switch to control the detrimental effects of microglia after acute brain injury, and provide a novel strategy to improve the outcome of ICH injury.

Conformationally Constrained Sialyl Analogues as New Potential Binders of h-CD22.

Here, two conformationally constrained sialyl analogues were synthesized and characterized in their interaction with the inhibitory Siglec, human CD22 (h-CD22). An orthogonal approach, including biophysical assays (SPR and fluorescence), ligand-based NMR techniques, and molecular modelling, was employed to disentangle the interaction mechanisms at a molecular level. The results showed that the Sialyl-TnThr antigen analogue represents a promising scaffold for the design of novel h-CD22 inhibitors. Our findings also suggest that the introduction of a biphenyl moiety at position 9 of the sialic acid hampers canonical accommodation of the ligand in the protein binding pocket, even though the affinity with respect to the natural ligand is increased. Our results address the search for novel modifications of the Neu5Ac-α(2-6)-Gal epitope, outline new insights for the design and synthesis of high-affinity h-CD22 ligands, and offer novel prospects for therapeutic intervention to prevent autoimmune diseases and B-cell malignancies.

Cutting Edge: ST8Sia6-Generated α-2,8-Disialic Acids Mitigate Hyperglycemia in Multiple Low-Dose Streptozotocin-Induced Diabetes.

The immune system contains a series of checks and balances that maintain tolerance and prevent autoimmunity. Sialic acid-binding Ig-type lectins (Siglecs) are cell surface receptors found on immune cells and inhibit inflammation by recruiting protein tyrosine phosphatases to ITIMs. Islet-resident macrophages express Siglec-E, and Siglec-E expression decreases on islet-resident macrophages as insulitis progresses in the NOD mouse. The sialyltransferase ST8Sia6 generates α-2,8-disialic acids that are ligands for Siglec-E in vivo. We hypothesized that engaging Siglec-E through ST8Sia6-generated ligands may inhibit the development of immune-mediated diabetes. Constitutive overexpression of ST8Sia6 in pancreatic ß cells mitigated hyperglycemia in the multiple low-dose streptozotocin model of diabetes, demonstrating that engagement of this immune receptor facilitates tolerance in the setting of inflammation and autoimmune disease.

The red blood cell as a novel regulator of human B-cell activation.

Non-immune cells are increasingly recognized as important in regulating immunity, but the role of red blood cells (RBC) remains relatively unexplored, despite their abundance in the circulation and a cell surface rich in potential ligands. Here, we determine whether RBC influence the activation state of human B cells. Separation of RBC from peripheral blood mononuclear cells increased B-cell expression of HLA-DR/DP/DQ, whilst reconstitution reduced the levels of B-cell activation markers HLA-DR/DP/DQ, CD86, CD69 and CD40, as well as decreasing proliferative responses and IgM secretion. Inhibition of B cells required contact with RBC and was abrogated by either removal of sialic acids from RBC or blocking the corresponding lectin receptor CD22 on B cells. Chronic lymphocytic leukaemia B cells express low levels of CD22 and were less susceptible to inhibition by RBC, which may contribute to their activated phenotype. Taken together, the results identify a novel mechanism that may suppress inappropriate responsiveness of healthy B cells whilst circulating in the bloodstream.

Glycoengineering of NK Cells with Glycan Ligands of CD22 and Selectins for B-Cell Lymphoma Therapy.

CD22, a member of Siglec family of sialic acid binding proteins, has restricted expression on B cells. Antibody-based agents targeting CD22 or CD20 on B lymphoma and leukemia cells exhibit clinical efficacy for treating these malignancies, but also attack normal B cells leading to immune deficiency. Here, we report a chemoenzymatic glycocalyx editing strategy to introduce high-affinity and specific CD22 ligands onto NK-92MI and cytokine-induced natural killer cells to achieve tumor-specific CD22 targeting. These CD22-ligand modified cells exhibited significantly enhanced tumor cell binding and killing in vitro without harming healthy B cells. For effective lymphoma cell killing in vivo, we further functionalized CD22 ligand-modified NK-92MI cells with the E-selectin ligand sialyl Lewis X to promote trafficking to bone marrow. The dual-functionalized cells resulted in the efficient suppression of B lymphoma in a xenograft model. Our results suggest that natural killer cells modified with glycan ligands to CD22 and selectins promote both targeted killing of B lymphoma cells and improved trafficking to sites where the cancer cells reside, respectively.

Nanoscale organization and dynamics of the siglec CD22 cooperate with the cytoskeleton in restraining BCR signalling.

Receptor organization and dynamics at the cell membrane are important factors of signal transduction regulation. Using super-resolution microscopy and single-particle tracking, we show how the negative coreceptor CD22 works with the cortical cytoskeleton in restraining BCR signalling. In naïve B cells, we found endogenous CD22 to be highly mobile and organized into nanodomains. The landscape of CD22 and its lateral diffusion were perturbed either in the absence of CD45 or when the CD22 lectin domain was mutated. To understand how a relatively low number of CD22 molecules can keep BCR signalling in check, we generated Brownian dynamic simulations and supported them with ex vivo experiments. This combined approach suggests that the inhibitory function of CD22 is influenced by its nanoscale organization and is ensured by its fast diffusion enabling a "global BCR surveillance" at the plasma membrane.

Mass Spectrometry-Based Shotgun Glycomics for Discovery of Natural Ligands of Glycan-Binding Proteins.

Glycans attached to lipids and membrane-bound and secreted proteins and peptides mediate many important physiological and pathophysiological processes through interactions with glycan-binding proteins (GBPs). However, uncovering functional glycan ligands is challenging due to the large number of naturally occurring glycan structures, the limited availability of glycans in their purified form, the low affinities of GBP-glycan interactions, and limitations in existing binding assays. This work explores the application of catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) for screening libraries of N-glycans derived from natural sources. The assay was tested by screening a small-defined library of complex N-glycans at equimolar concentrations against plant and human GBPs with known specificities for either α2-3- or α2-6-linked sialosides, with affinities in the millimolar to micromolar range. Validation experiments, performed in negative ion mode, revealed that bound N-glycan ligands are readily released, as intact deprotonated ions, from GBPs in the gas phase using collision-induced dissociation. Moreover, the relative abundances of the released ligands closely match their solution affinities. The results obtained for a natural N-glycan library produced from cultured immune cells serve to highlight the ease with which CaR-ESI-MS can screen complex mixtures of N-glycans for interactions. Additionally, scaling the relative abundances of released glycan ligands according to their relative abundances in solution, as determined by hydrophilic interaction-ultrahigh-performance liquid chromatography of the fluorescently labeled library, allows the relative affinities of glycan ligands to be ranked.

Efficacy and toxicity for CD22/CD19 chimeric antigen receptor T-cell therapy in patients with relapsed/refractory aggressive B-cell lymphoma involving the gastrointestinal tract.

Gastrointestinal (GI) tract is the most common site of extranodal involvement in non-Hodgkin lymphoma. Life-threatening complications of GI may occur because of tumor or chemotherapy. Chimeric antigen receptor (CAR) T-cell therapy has been successfully used to treat refractory/relapse B-cell lymphoma, however, little is known about the efficacy and safety of CAR-T cell therapy for GI lymphoma. Here, we reported the efficacy and safety of CAR-T cell therapy in 14 patients with relapsed/refractory aggressive B-cell lymphoma involving the GI tract. After a sequential anti-CD22/anti-CD19 CAR-T therapy, 10 patients achieved an objective response, and seven patients achieved a complete response. CAR transgene and B-cell aplasia persisted in the majority of patients irrespective of response status. Six patients with partial response or stable disease developed progressive disease; two patients lost target antigens. Cytokine release syndrome (CRS) and GI adverse events were generally mild and manageable. The most common GI adverse events were diarrhea (4/14), vomiting (3/14) and hemorrhage (2/14). No perforation occurred during follow-up. Infection is a severe complication in GI lymphoma. Two patients were infected with bacteria that are able to colonize at GI; one died of sepsis early after CAR-T cells infusion. In conclusion, our study showed promising efficacy and safety of CAR-T cell therapy in refractory/relapsed B-cell lymphoma involving the GI tract. However, the characteristics of CAR-T-related infection in GI lymphoma should be further clarified to prevent and control infection.

Disruption of hepatocyte Sialylation drives a T cell-dependent pro-inflammatory immune tone.

Through the catalysis of α2,6-linked sialylation, the enzyme ST6Gal1 is thought to play key roles in immune cell communication and homeostasis. Of particular importance, glycans with terminal α2,6-sialic acids are known to negatively regulate B cell receptor signaling and are associated with an immunosuppressive tumor microenvironment that promotes T cell anergy, suggesting that α2,6-sialic acids are a key immune inhibitory signal. Consistent with this model, mice harboring a hepatocyte-specific ablation of ST6Gal1 (H-cKO) develop a progressive and severe non-alcoholic fatty liver disease characterized by steatohepatitis. Using this H-cKO mouse, we have further discovered that loss of hepatocyte α2,6-sialylation not only increases the inflammatory state of the local tissue microenvironment, but also systemic T cell-dependent immune responses. H-cKO mice responded normally to innate and passively induced inflammation, but showed significantly increased morbidity in T cell-dependent house dust mite-antigen (HDM)-induced asthma and myelin oligodendrocyte glycoprotein (MOG) peptide-induced experimental autoimmune encephalomyelitis (EAE). We further discovered that H-cKO mice have a profound shift toward effector/memory T cells even among unchallenged mice, and that macrophages from both the liver and spleen expressed the inhibitory and α2,6-sialic acid-specific glycan binding molecule CD22. These findings align with previously reported pro-inflammatory changes in liver macrophages, and support a model in which the liver microenvironment sets a systemic immune tone that is regulated by tissue α2,6-sialylation and mediated by liver macrophages and systemic T cells.

Bacterial expression and characterization of an anti-CD22 single-chain antibody fragment.

Single-chain variable fragment (scFv) antibodies are fusion proteins of the variable regions of the heavy and light chains of immunoglobulins connected with a short linker peptide. They possess unique and superior features compared to whole antibodies for immunotherapy of various carcinomas, including hematologic B-cell malignancies. In the presented study we obtained efficient production of the recombinant anti-CD22 scFv in Escherichia coli expression system. The active recombinant protein was successfully recovered from inclusion bodies. Assays were performed to assess the in vitro targeting properties and specificity of the obtained anti-CD22 scFv antibody in the CD22 positive and negative lymphoma cell lines.

Sialic Acid Ligand Binding of CD22 and Siglec-G Determines Distinct B Cell Functions but Is Dispensable for B Cell Tolerance Induction.

Siglec-G and CD22 are inhibitory receptors on B cells and play an important role in the maintenance of tolerance. Although both molecules are expressed on all B cell populations at a similar level, Siglec-G was found to regulate exclusively B1a cells, whereas CD22 functions as an inhibitory receptor specifically on B2 cells. It is known that the mechanistic function of both Siglecs is regulated by sialic acid binding in a reciprocal manner, although it was not known until now how B cells would act when both Siglec-G and CD22 lack their ability to bind sialic acids. We answered this question by analyzing Siglec-G R120E x CD22 R130E mice. These mice show decreased numbers of mature recirculating B cells in the bone marrow similar to mice with mutations in CD22. Also, they show an increased B1a cell population in peritoneal cavity and a skewed BCR repertoire in peritoneal B1a cells, which is characteristic for mice with mutated Siglec-G. Ca2+ mobilization was strongly reduced in B2 cells and was altered in peritoneal B1a cells, whereas B cell survival was neither affected in B2 cells nor in B1a cells. Also, aging Siglec-G R120E x CD22 R130E mice do neither develop a general hyperactivated immune status nor autoimmunity. This demonstrates that Siglec binding to sialic acids as abundant self-ligands cannot be a dominant mechanism for the Siglec-mediated B cell tolerance induction.

Sialic acids and autoimmune disease.

An important underlying mechanism that contributes to autoimmunity is the loss of inhibitory signaling in the immune system. Sialic acid-recognizing Ig superfamily lectins or Siglecs are a family of cell surface proteins largely expressed in hematopoietic cells. The majority of Siglecs are inhibitory receptors expressed in immune cells that bind to sialic acid-containing ligands and recruit SH2-domain-containing tyrosine phosphatases to their cytoplasmic tails. They deliver inhibitory signals that can contribute to the constraining of immune cells, and thus protect the host from autoimmunity. The inhibitory functions of CD22/Siglec-2 and Siglec-G and their contributions to tolerance and autoimmunity, primarily in the B lymphocyte context, are considered in some detail in this review. The relevance to autoimmunity and unregulated inflammation of modified sialic acids, enzymes that modify sialic acid, and other sialic acid-binding proteins are also reviewed.

Exposure-Efficacy Analysis of Antibody-Drug Conjugates Delivering an Excessive Level of Payload to Tissues.

Antibody-drug conjugates (ADCs) contain a disease-receptor antibody and a payload drug connected via a linker. The payload delivery depends on both tumor properties and ADC characteristics. In this study, we used different linkers, attachment sites, and doses to modulate payload delivery of several ADCs bearing maytansinoids (e.g., DM1), auristatins (e.g., MMAE), and DNA alkylating agents [e.g., pyrrolo[2,1-c][1,4]benzodiazepine-dimer (PBD)] as payloads in HER2- or CD22-expressing xenograft models. The tumor growth inhibition and ADC stability and exposure data were collected and analyzed from these dosed animals. The trend analysis suggests that intratumoral payload exposures that directly related the combination of conjugate linker and dose correlate with the corresponding efficacies of three payload types in two antigen-expressing xenograft models. These preliminary correlations also suggest that a minimal threshold concentration of intratumoral payload is required to support sustained efficacy. In addition, an ADC can deliver an excessive level of payload to tumors that does not enhance efficacy ("Plateau" effect). In contrast to tumor payload concentrations, the assessments of systemic exposures of total antibody (Tab) as well as the linker, dose, site of attachment, plasma stability, and drug-to-antibody ratio changes of these ADCs did not consistently rationalize the observed ADC efficacies. The requirement of a threshold payload concentration for efficacy is further supported by dose fractionation studies with DM1-, MMAE-, and PBD-containing ADCs, which demonstrated that single-dose regimens showed better efficacies than fractionated dosing. Overall, this study demonstrates that 1) the linker and dose together determine the tissue payload concentration that correlates with the antitumor efficacy of ADCs and 2) an ADC can deliver an unnecessary level of payload to tumors in xenograft models.