Enhancement of complement-dependent cytotoxicity by linking factor-H derived short consensus repeats 19-20 to CD20 antibodies.

Antibody-mediated complement-dependent cytotoxicity (CDC) on malignant cells is regulated by several complement control proteins, including the inhibitory complement factor H (fH). fH consists of 20 short consensus repeat elements (SCRs) with specific functional domains. Previous research revealed that the fH-derived SCRs 19-20 (SCR1920) can displace full-length fH on the surface of chronic lymphocytic leukemia (CLL) cells, which sensitizes CLL cells for e.g. CD20-targeting therapeutic monoclonal antibody (mAb) induced CDC. Therefore, we constructed lentiviral vectors for the generation of cell lines that stably produce mAb-SCR-fusion variants starting from the clinically approved parental mAbs rituximab, obinutuzumab and ofatumumab, respectively. Flow-cytometry revealed that the modification of the mAbs by the SCRs does not impair the binding to CD20. Increased in vitro lysis potency compared to their parental mAbs was corroborated by showing specific and dose dependent target cell elimination by CDC when compared to their parental mAbs. Lysis of CLL cells was not affected by the depletion of NK cells, suggesting that antibody-dependent cellular cytotoxicity plays a minor role in this context. Overall, this study emphasizes the crucial role of CDC in the elimination of CLL cells by mAbs and introduces a novel approach for enhancing CDC by directly fusing fH SCR1920 with mAbs.

Prevalence of anti-lymphocyte IgM autoantibodies driving complement activation in COVID-19 patients.

Introduction: COVID-19 patients can develop autoantibodies against a variety of secreted and membrane proteins, including some expressed on lymphocytes. However, it is unclear what proportion of patients might develop anti-lymphocyte antibodies (ALAb) and what functional relevance they might have. Methods: We evaluated the presence and lytic function of ALAb in the sera of a cohort of 85 COVID-19 patients (68 unvaccinated and 17 vaccinated) assigned to mild (N=63), or moderate/severe disease (N=22) groups. Thirty-seven patients were followed-up after recovery. We also analyzed in vivo complement deposition on COVID-19 patients' lymphocytes and examined its correlation with lymphocyte numbers during acute disease. Results: Compared with healthy donors (HD), patients had an increased prevalence of IgM ALAb, which was significantly higher in moderate/severe disease patients and persisted after recovery. Sera from IgM ALAb+ patients exhibited complement-dependent cytotoxicity (CDC) against HD lymphocytes. Complement protein C3b deposition on patients' CD4 T cells was inversely correlated with CD4 T cell numbers. This correlation was stronger in moderate/severe disease patients. Discussion: IgM ALAb and complement activation against lymphocytes may contribute to the acute lymphopenia observed in COVID-19 patients.

Maitake Beta-Glucan Enhances the Therapeutic Effect of Trastuzumab via Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity.

Trastuzumab, an anti-HER2 monoclonal antibody, is the mainstay treatment for of HER2-positive breast cancer. However, trastuzumab resistance is often observed during treatment. Therefore, new therapeutic strategies are needed to enhance the clinical benefits of trastuzumab. Maitake ß-glucan MD-Fraction, isolated from Grifola frondosa, inhibits tumor growth by enhancing immune responses. In this study, we examined the effect of MD-Fraction on trastuzumab treatment of HER2-positive breast cancer. MD-Fraction did not directly inhibit the survival of HER2-positive breast cancer cells, alone or in the presence of trastuzumab in vitro. In HER2-positive xenograft models, the combination of MD-Fraction and trastuzumab was more effective than trastuzumab alone. Peripheral blood lymphocytes and splenic natural killer cells isolated from BALB/c nu/nu mice treated with MD-Fraction showed enhanced trastuzumab-induced antibody-dependent cellular cytotoxicity (ADCC) ex vivo. MD-Fraction-treated macrophages and neutrophils did not show enhanced trastuzumab cytotoxicity in the presence of heat-inactivated serum, but they showed enhanced cytotoxicity in the presence of native serum. These results suggest that MD-Fraction-treated macrophages and neutrophils enhance trastuzumab-induced complement-dependent cellular cytotoxicity (CDCC). Treatment of HER2-positive breast cancer cells with MD-Fraction in the presence of trastuzumab and native serum increased C3a release and tumor cell lysis in a dose-dependent manner, indicating that MD-Fraction enhanced trastuzumab-induced complement-dependent cytotoxicity (CDC) by activating the complement system. This study demonstrates that the combination of trastuzumab and MD-Fraction exerts a greater antitumor effect than trastuzumab alone by enhancing ADCC, CDCC, and CDC in HER2-positive breast cancer.

CD46 and CD59 inhibitors enhance complement-dependent cytotoxicity of anti-CD38 monoclonal antibodies daratumumab and isatuximab in multiple myeloma and other B-cell malignancy cells.

Multiple myeloma (MM) is an incurable malignancy of the B-cell lineage. Remarkable progress has been made in the treatment of MM with anti-CD38 monoclonal antibodies such as daratumumab and isatuximab, which can kill MM cells by inducing complement-dependent cytotoxicity (CDC). We showed that the CDC efficacy of daratumumab and isatuximab is limited by membrane complement inhibitors, including CD46 and CD59, which are upregulated in MM cells. We recently developed a small recombinant protein, Ad35K++, which is capable of transiently removing CD46 from the cell surface. We also produced a peptide inhibitor of CD59 (rILYd4). In this study, we tested Ad35K++ and rILYd4 in combination with daratumumab and isatuximab in MM cells as well as in cells from two other B-cell malignancies. We showed that Ad35K++ and rILYd4 increased CDC triggered by daratumumab and isatuximab. The combination of both inhibitors had an additive effect in vitro in primary MM cells as well as in vivo in a mouse xenograft model of MM. Daratumumab and isatuximab treatment of MM lines (without Ad35K++ or rILYd4) resulted in the upregulation of CD46/CD59 and/or survival of CD46high/CD59high MM cells that escaped the second round of daratumumab and isatuximab treatment. The escape in the second treatment cycle was prevented by the pretreatment of cells with Ad35K++. Overall, our data demonstrate that Ad35K++ and rILYd4 are efficient co-therapeutics of daratumumab and isatuximab, specifically in multi-cycle treatment regimens, and could be used to improve treatment of multiple myeloma.

Therapeutic efficacy of a potent anti-Venezuelan equine encephalitis virus antibody is contingent on Fc effector function.

The development of specific, safe, and potent monoclonal antibodies (Abs) has led to novel therapeutic options for infectious disease. In addition to preventing viral infection through neutralization, Abs can clear infected cells and induce immunomodulatory functions through engagement of their crystallizable fragment (Fc) with complement proteins and Fc receptors on immune cells. Little is known about the role of Fc effector functions of neutralizing Abs in the context of encephalitic alphavirus infection. To determine the role of Fc effector function in therapeutic efficacy against Venezuelan equine encephalitis virus (VEEV), we compared the potently neutralizing anti-VEEV human IgG F5 (hF5) Ab with intact Fc function (hF5-WT) or containing the loss of function Fc mutations L234A and L235A (hF5-LALA) in the context of VEEV infection. We observed significantly reduced binding to complement and Fc receptors, as well as differential in vitro kinetics of Fc-mediated cytotoxicity for hF5-LALA compared to hF5-WT. The in vivo efficacy of hF5-LALA was comparable to hF5-WT at -24 and + 24 h post infection, with both Abs providing high levels of protection. However, when hF5-WT and hF5-LALA were administered + 48 h post infection, there was a significant decrease in the therapeutic efficacy of hF5-LALA. Together these results demonstrate that optimal therapeutic Ab treatment of VEEV, and possibly other encephalitic alphaviruses, requires neutralization paired with engagement of immune effectors via the Fc region.

A Humanized and Defucosylated Antibody against Podoplanin (humLpMab-23-f) Exerts Antitumor Activities in Human Lung Cancer and Glioblastoma Xenograft Models.

A cancer-specific anti-PDPN mAb, LpMab-23 (mouse IgG1, kappa), was established in our previous study. We herein produced a humanized IgG1 version (humLpMab-23) and defucosylated form (humLpMab-23-f) of an anti-PDPN mAb to increase ADCC activity. humLpMab-23 recognized PDPN-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/PDPN), PDPN-positive PC-10 (human lung squamous cell carcinoma), and LN319 (human glioblastoma) cells via flow cytometry. We then demonstrated that humLpMab-23-f induced ADCC and complement-dependent cytotoxicity against CHO/PDPN, PC-10, and LN319 cells in vitro and exerted high antitumor activity in mouse xenograft models, indicating that humLpMab-23-f could be useful as an antibody therapy against PDPN-positive lung squamous cell carcinomas and glioblastomas.

Assessment of mimicking by EBV-CMV immunoglobulin M of anti-HLA antibodies.

OBJECTIVE: We aimed to show the cross-reactivity that may occur between immunoglobulin (Ig) M antibodies that form against Cytomegalovirus (CMV) and/or Epstein-Barr virus (EBV) and human leukocyte antigens (HLA). METHODS: Complement-dependent cytotoxicity (CDC) cross-reactivity between serum samples of 57 patients with IgM positive CMV and/or EBV infections and T and B cells from 15 healthy donors were evaluated. Dithiothreitol was used to distinguish cross-reactivity caused by IgM antibodies from IgG. RESULTS: The cross-reactivity ratio between pathogenic IgM antibodies with T cell of the 12th donor, and B cell of the 3rd, 4th, and 8th donors was significantly higher (p = 0.011, <0.001, <0.001 and 0.013, respectively). The ratio of B cell CDC cross-reactivity of all donors (26.4%) was higher than the ratio of T cell CDC cross-reactivity (5.2%) (p < 0.001). The ratio of T cell CDC cross-reactivity of sera containing both anti-CMV IgM and anti-EBV IgM antibodies was significantly higher than those of sera containing only anti-CMV IgM or only anti-EBV IgM antibodies (p = 0.002 and p < 0.001, respectively). There was no difference between B cell CDC cross-reactivity rates according to the presence of anti-CMV and/or anti-EBV IgM antibodies. CONCLUSION: Cross-reactivity may occur between anti-CMV and anti-EBV IgM antibodies with HLA molecules. Thus, in graft recipients, pathogenic IgMs can also act as de novo anti-HLA antibodies and aggravate the rejection process.

The role of N-glycosylation in B-cell biology and IgG activity. The aspects of autoimmunity and anti-inflammatory therapy.

The immune system is strictly regulated by glycosylation through the addition of highly diverse and dynamically changing sugar structures (glycans) to the majority of immune cell receptors. Although knowledge in the field of glycoimmunology is still limited, numerous studies point to the key role of glycosylation in maintaining homeostasis, but also in reflecting its disruption. Changes in oligosaccharide patterns can lead to impairment of both innate and acquired immune responses, with important implications in the pathogenesis of diseases, including autoimmunity. B cells appear to be unique within the immune system, since they exhibit both innate and adaptive immune activity. B cell surface is rich in glycosylated proteins and lectins which recognise glycosylated ligands on other cells. Glycans are important in the development, selection, and maturation of B cells. Changes in sialylation and fucosylation of cell surface proteins affect B cell signal transduction through BCRs, CD22 inhibitory coreceptor and Siglec-G. Plasmocytes, as the final stage of B cell differentiation, produce and secrete immunoglobulins (Igs), of which IgGs are the most abundant N-glycosylated proteins in human serum with the conserved N-glycosylation site at Asn297. N-oligosaccharide composition of the IgG Fc region affects its secretion, structure, half-life and effector functions (ADCC, CDC). IgG N-glycosylation undergoes little change during homeostasis, and may gradually be modified with age and during ongoing inflammatory processes. Hyperactivated B lymphocytes secrete autoreactive antibodies responsible for the development of autoimmunity. The altered profile of IgG N-glycans contributes to disease progression and remission and is sensitive to the application of therapeutic substances and immunosuppressive agents. In this review, we focus on the role of N-glycans in B-cell biology and IgG activity, the rearrangement of IgG oligosaccharides in aging, autoimmunity and immunosuppressive therapy.

Preclinical anti-tumour activity of HexaBody-CD38, a next-generation CD38 antibody with superior complement-dependent cytotoxic activity.

BACKGROUND: HexaBody®-CD38 (GEN3014) is a hexamerization-enhanced human IgG1 that binds CD38 with high affinity. The E430G mutation in its Fc domain facilitates the natural process of antibody hexamer formation upon binding to the cell surface, resulting in increased binding of C1q and potentiated complement-dependent cytotoxicity (CDC). METHODS: Co-crystallization studies were performed to identify the binding interface of HexaBody-CD38 and CD38. HexaBody-CD38-induced CDC, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), trogocytosis, and apoptosis were assessed using flow cytometry assays using tumour cell lines, and MM patient samples (CDC). CD38 enzymatic activity was measured using fluorescence spectroscopy. Anti-tumour activity of HexaBody-CD38 was assessed in patient-derived xenograft mouse models in vivo. FINDINGS: HexaBody-CD38 binds a unique epitope on CD38 and induced potent CDC in multiple myeloma (MM), acute myeloid leukaemia (AML), and B-cell non-Hodgkin lymphoma (B-NHL) cells. Anti-tumour activity was confirmed in patient-derived xenograft models in vivo. Sensitivity to HexaBody-CD38 correlated with CD38 expression level and was inversely correlated with expression of complement regulatory proteins. Compared to daratumumab, HexaBody-CD38 showed enhanced CDC in cell lines with lower levels of CD38 expression, without increasing lysis of healthy leukocytes. More effective CDC was also confirmed in primary MM cells. Furthermore, HexaBody-CD38 efficiently induced ADCC, ADCP, trogocytosis, and apoptosis after Fc-crosslinking. Moreover, HexaBody-CD38 strongly inhibited CD38 cyclase activity, which is hypothesized to relieve immune suppression in the tumour microenvironment. INTERPRETATION: Based on these preclinical studies, a clinical trial was initiated to assess the clinical safety of HexaBody-CD38 in patients with MM. FUNDING: Genmab.

Complement contributes to antibody-mediated protection against repeated SHIV challenge.

The first clinical efficacy trials of a broadly neutralizing antibody (bNAb) resulted in less benefit than expected and suggested that improvements are needed to prevent HIV infection. While considerable effort has focused on optimizing neutralization breadth and potency, it remains unclear whether augmenting the effector functions elicited by broadly neutralizing antibodies (bNAbs) may also improve their clinical potential. Among these effector functions, complement-mediated activities, which can culminate in the lysis of virions or infected cells, have been the least well studied. Here, functionally modified variants of the second-generation bNAb 10-1074 with ablated and enhanced complement activation profiles were used to examine the role of complement-associated effector functions. When administered prophylactically against simian-HIV challenge in rhesus macaques, more bNAb was required to prevent plasma viremia when complement activity was eliminated. Conversely, less bNAb was required to protect animals from plasma viremia when complement activity was enhanced. These results suggest that complement-mediated effector functions contribute to in vivo antiviral activity, and that their engineering may contribute to the further improvements in the efficacy of antibody-mediated prevention strategies.

Functional Validation of the RQR8 Suicide /Marker Gene in CD19 CAR-T Cells and CLL1CAR-T Cells.

Chimeric antigen receptor T cell therapy (CAR-T) is a novel treatment that has produced unprecedented clinical effects in patients with hematological malignancies. Acute adverse events often occur following adoptive immunotherapy. Therefore, a suicide gene is helpful, which is a genetically encoded mechanism that allows selective destruction of adoptively transferred T cells in the face of unacceptable toxicity. RQR8 is a gene that integrates CD34 and CD20 epitopes. In our study, we incorporated the suicide gene RQR8 into CAR-T cells, so it enabled rituximab to eliminate vector/transgene-expressing T cells via antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity. In this work, we explored the functionality of RQR8 CAR-T cells in vitro and in vivo. We believe that RQR8 as a safety switch will make CAR-T cell therapy safer and less costly.

Improvement of Antibody Activity by Controlling Its Dynamics Using the Glycan-Lectin Interaction.

Antibody dynamics on membranes, such as endocytosis and clustering, are vital in determining antibody functions. In this study, we demonstrated that glycan conjugation can modulate antibody dynamics through the glycan-lectin interaction to regulate its potency. The anti-HER2 antibody, an anti-breast-cancer antibody, was conjugated with galactose-containing N-glycan, and its internalization was suppressed by interaction with galectin-3, leading to enhanced complement-dependent cytotoxic (CDC) activity. This glycan-antibody conjugate is proposed as a new approach to modulate antibody activity and may provide an alternative strategy for redeveloping antibody drugs that do not exhibit sufficient activity.

Detection of donor-specific HLA antibodies: A retrospective observation in 350 renal transplant cases.

BACKGROUND: The main objective of this study was to determine the results of the cell-based assay (CDC-XM and FC-XM), and correlate with the results of solid phase assay (L-SAB). METHODS: In this retrospective study, 350 prospective renal transplant recipients were tested for the presence of HLA antibodies by CDC-XM, FC-XM and L-SAB screening with their corresponding donor. RESULTS: T-cell-FC-XM showed a sensitivity of 71.43% and a specificity of 91.50% for detecting class I L-SAB (+), while B-cell-FCXM showed a sensitivity of 94.94% and a specificity of 61.99% for detecting class II L-SAB (+). On the other hand, T-CDC-XM showed a sensitivity of 32.14% and a specificity of 98.64% for detecting class I L-SAB (+), while B-CDC-XM showed a sensitivity of 44.30% and a specificity of 94.83% for detecting class II L-SAB (+). In this study, the results indicated that DSA class I MFI value of 2845 and above significantly (p ≤0.001) correlated with T-cell-FC-XM positivity, while MFI value of 4585 and above (p ≤0.001) showed strong predictive accuracy of a positive T-cell-CDC-XM. However, DSA class II MFI cut-off of 1988 and above significantly (p ≤0.001) correlated with B-cell-FC-XM positivity, while MFI value of 5986 and above (p ≤0.001) showed strong predictive accuracy of a positive B-cell-CDC-XM. CONCLUSIONS: Our study showed that CDC-XM has poor sensitivity, while FC-XM has poor specificity to detect DSA. L-SAB has good correlation with T-cell-FC-XM (p < 0.0001) but not with B-cell-FC-XM (P = 0.31). DSA strength >2845 and > 1988 significantly correlated with T-cell-FC-XM positivity and B-cell-FC-XM positivity, respectively. While, a MFI value of >4585 and > 5986 significantly correlated with T-cell-CDC-XM positivity and B-cell-CDC-XM positivity, respectively. These MFI cut-off values could serve as a surrogate marker for CDC-XM and FC-XM tests and may help in resolving the limitations of cell-based techniques. In conclusion, we found that L-SAB is more sensitive and specific than CDC-XM and FC-XM and therefore may be used as a test of choice.

Daratumumab induces cell-mediated cytotoxicity of primary effusion lymphoma and is active against refractory disease.

Primary effusion lymphoma (PEL), an aggressive non-Hodgkin lymphoma caused by Kaposi sarcoma-associated herpesvirus (KSHV), lacks standard therapy and has a median survival of 10-22 months with combination chemotherapy. PEL is a tumor of plasmablast-like B cells generally expressing CD38, the target of daratumumab (Dara). Initially, we assessed PEL cells from eight patients and established that each expressed high levels of CD38 by flow cytometry. PEL cell lines were also evaluated and most had high CD38 expression. We then assessed Dara's effects on complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) of PEL cell lines as well as its clinical benefits on two patients with PEL. Despite high CD38 expression, Dara did not induce CDC of PEL cell lines, due in part to high levels of the complement-inhibitory proteins, CD55 and CD59. However, Dara induced significant and dose-dependent increases in ADCC, particularly in those lines with high CD38 levels. Two FDA-approved drugs, all trans-retinoic acid (ATRA) and pomalidomide (Pom), significantly increased surface CD38 levels in low-CD38 expressing PEL cell lines, resulting in increased Dara-induced ADCC. Two patients with refractory PEL were treated with Dara alone or in combination with Pom. One patient with leptomeningeal PEL had a complete response to Dara and Pom combination treatment. Others had improvement in performance status and resolution of malignant ascites with Dara alone. Together, these data support the use of Dara monotherapy or in combination with ATRA or Pom as a potential therapeutic option for PEL.

Antitumor Activities in Mouse Xenograft Models of Canine Fibroblastic Tumor by Defucosylated Mouse-Dog Chimeric Anti-HER2 Monoclonal Antibody (H77Bf).

Human epidermal growth factor receptor 2 (HER2) is a cell surface type I transmembrane glycoprotein that is overexpressed on a variety of solid tumors and transduces the oncogenic signaling upon homo- and heterodimerization with HER families. Anti-HER2 monoclonal antibodies (mAbs) including trastuzumab and its antibody-drug conjugate have been shown to improve patients' survival in HER2-positive breast, gastric, and lung cancers. Canine tumors have advantages as naturally occurring tumor models, and share biological and histological characteristics with human tumors. In this study, we generated a defucosylated version of mouse-dog chimeric anti-HER2 mAb (H77Bf) derived from H2Mab-77 (mouse IgG1, kappa). H77Bf possesses the high binding affinity (a dissociation constant: 8.7 × 10-10 M) for a dog HER2 (dHER2)-expressing canine fibroblastic tumor cell line (A-72). H77Bf exhibited antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity for A-72 cells. Moreover, intraperitoneal administration of H77Bf significantly suppressed the development of A-72 tumor compared with the control dog IgG in a mouse xenograft model. These results indicate that H77Bf exerts antitumor activities against dHER2-expressing canine cancers, which could provide a valuable information for canine cancer treatment.

miR-132-3p regulates antibody-mediated complement-dependent cytotoxicity in colon cancer cells by directly targeting CD55.

The overexpression of membrane-bound complement regulatory proteins (mCRPs) on tumour cells helps them survive complement attacks by suppressing antibody-mediated complement-dependent cytotoxicity (CDC). Consequently, mCRP overexpression limits monoclonal antibody drug immune efficacy. CD55, an mCRP, plays an important role in inhibiting antibody-mediated CDC. However, the mechanisms regulating CD55 expression in tumour cells remain unclear. Here, the aim was to explore CD55-targeting miRNAs. We previously constructed an in vitro model comprising cancer cell lines expressing α-gal and serum containing natural antibodies against α-gal and complement. This was used to simulate antibody-mediated CDC in colon cancer cells. We screened microRNAs that directly target CD55 using LoVo and Ls-174T colon cell lines, which express CD55 at low and high levels, respectively. miR-132-3p expression was dramatically lower in Ls-174T cells than in LoVo cells. miR-132-3p overexpression or inhibition transcriptionally regulated CD55 expression by specifically targeting its mRNA 3'-untranslated regions. Further, miR-132-3p modulation regulated colon cancer cell sensitivity to antibody-mediated CDC through C5a release and C5b-9 deposition. Moreover, miR-132-3p expression was significantly reduced, whereas CD55 expression was increased, in colon cancer tissues compared to levels in adjacent normal tissues. CD55 protein levels were negatively correlated with miR-132-3p expression in colon cancer tissues. Our results indicate that miR-132-3p regulates colon cancer cell sensitivity to antibody-mediated CDC by directly targeting CD55. In addition, incubating the LoVo human tumour cell line, stably transfected with the xenoantigen α-gal, with human serum containing natural antibodies comprises a stable and cheap in vitro model to explore the mechanisms underlying antibody-mediated CDC.

Single domain antibodies derived from ancient animals as broadly neutralizing agents for SARS-CoV-2 and other coronaviruses.

With severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as an emergent human virus since December 2019, the world population is susceptible to coronavirus disease 2019 (COVID-19). SARS-CoV-2 has higher transmissibility than the previous coronaviruses, associated by the ribonucleic acid (RNA) virus nature with high mutation rate, caused SARS-CoV-2 variants to arise while circulating worldwide. Neutralizing antibodies are identified as immediate and direct-acting therapeutic against COVID-19. Single-domain antibodies (sdAbs), as small biomolecules with non-complex structure and intrinsic stability, can acquire antigen-binding capabilities comparable to conventional antibodies, which serve as an attractive neutralizing solution. SARS-CoV-2 spike protein attaches to human angiotensin-converting enzyme 2 (ACE2) receptor on lung epithelial cells to initiate viral infection, serves as potential therapeutic target. sdAbs have shown broad neutralization towards SARS-CoV-2 with various mutations, effectively stop and prevent infection while efficiently block mutational escape. In addition, sdAbs can be developed into multivalent antibodies or inhaled biotherapeutics against COVID-19.

Clinical features of organizing pneumonia in anti-aquaporin-4 antibody-positive neuromyelitis optica spectrum disorders.

BACKGROUND: Anti-aquaporin-4 (AQP4) antibody is an autoantibody marker often observed in patients with neuromyelitis optica spectrum disorder (NMOSD). The pathological relevance of complicated pulmonary disorders in anti-AQP4 antibody-positive NMOSD remains unclear. We aimed to assess the clinical and histological relevance of complicated pulmonary disorders in anti-AQP4 antibody-positive NMOSD. METHODS: We retrospectively reviewed the medical records of 52 patients with anti-AQP4 antibody-positive NMOSD and conducted immunohistochemical evaluations of the lung biopsy specimens. RESULTS: Among 52 patients with anti-AQP4 antibody-positive NMOSD, 4 patients showed pulmonary involvement with a diagnosis of organizing pneumonia (OP). The proportion of males was larger (75% vs. 12.5%; p = 0.013) and creatine kinase levels were higher (458.3 U/L vs. 83.9 U/L; p = 0.003) in patients with OP than in those without OP. OP development preceded or coincided with the NMOSD symptoms. Chest computed tomography findings were consistent with OP in all four patients. Bronchoalveolar lavage fluid predominantly contained lymphocytes. Transbronchial lung biopsy revealed intraluminal plugs of inflammatory debris within the alveoli. Alveolar epithelial cells covering the OP lesions exhibited AQP4 loss, immunoglobulin G deposition, and complement activation. Corticosteroid treatment resulted in clinical improvement of OP. CONCLUSION: OP may be considered a pulmonary manifestation of anti-AQP4 antibody-positive NMOSD beyond the central nervous system. Complement-dependent cytotoxicity of the lung epithelial cells caused by anti-AQP4 antibody is at least partly involved in OP development. When diagnosing NMOSD, the possibility of OP should be carefully evaluated based on the detailed history and chest imaging findings.

Antibody-mediated depletion of programmed death 1-positive (PD-1+) cells.

PD-1 immune checkpoint has been intensively investigated in pathogenesis and treatments for cancer and autoimmune diseases. Cells that express PD-1 (PD-1+ cells) draw ever-increasing attention in cancer and autoimmune disease research although the role of PD-1+ cells in the progression and treatments of these diseases remains largely ambiguous. One definite approach to elucidate their roles is to deplete these cells in disease settings and examine how the depletion impacts disease progression and treatments. To execute the depletion, we designed and generated the first depleting antibody (D-αPD-1) that specifically ablates PD-1+ cells. D-αPD-1 has the same variable domains as an anti-mouse PD-1 blocking antibody (RMP1-14). The constant domains of D-αPD-1 were derived from mouse IgG2a heavy and κ-light chain, respectively. D-αPD-1 was verified to bind with mouse PD-1 as well as mouse FcγRIV, an immuno-activating Fc receptor. The cell depletion effect of D-αPD-1 was confirmed in vivo using a PD-1+ cell transferring model. Since transferred PD-1+ cells, EL4 cells, are tumorigenic and EL4 tumors are lethal to host mice, the depleting effect of D-αPD-1 was also manifested by an absolute survival among the antibody-treated mice while groups receiving control treatments had median survival time of merely approximately 30 days. Furthermore, we found that D-αPD-1 leads to elimination of PD-1+ cells through antibody-dependent cell-mediate phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) mechanisms. These results, altogether, confirmed the specificity and effectiveness of D-αPD-1. The results also highlighted that D-αPD-1 is a robust tool to study PD-1+ cells in cancer and autoimmune diseases and a potential therapeutic for these diseases.

Effects of different sensitization events on HLA alloimmunization in renal transplant cases; a retrospective observation in 1066 cases.

BACKGROUND: Patients awaiting solid organ transplantation may develop anti-HLA antibodies after sensitization events such as transfusions, pregnancies, or previous transplantations. However, the effects of a particular sensitization event on HLA alloimmunization have not been well studied in parallel using cell-based assays and solid-phase assays. In this study, we evaluated and compare how different sensitization events affect the HLA antibody screening (HLA-Ab) and donor specific antibody (DSA) status in solid renal organ transplantation patients. METHODS: HLA antibody (HLA-Ab) screening tests like complement-dependent cytotoxicity crossmatch (CDC-XM), flow cytometry crossmatch (FC-XM) and Luminex panel-reactive antibody (L-PRA) were performed in all 1066 patients (635 males and 431 females). If any of these tests turned out to be positive, a Luminex single antigen bead (L-SAB) assay was performed for DSA identification. Test positive rates and antibody strengths were analyzed according to the different sensitization events and gender. RESULTS: In this study, HLA-Ab screening tests positive rates (L-PRA, FC-XM and CDC-XM) were significantly higher in patients with previous transplantation (73.91%, 100% and 56.52% p < 0.001), previous pregnancy (57.46%, 70.14% and 18.85% p < 0.001) or blood transfusion (27.33%, 35.55% and 7.33% p < 0.001) compared with patients without a sensitizing event (6.17%, 13.58% and 1.09). In this study, re-transplantation group showed significantly stronger antibody strength (DSA) than non sensitized group (class I and II MFI 11418.04, 17,837.78 vs class I and II MFI 2659, 3329; P < 0.001) and those with single sensitization events of transfusion (class I and II MFI 11418.04, 17,837.78 vs class I and II MFI 5790.26, 6004.16; P < 0.001) or pregnancy (class I & II MFI 11418, 17,837 vs class I and II MFI 8631.71, 7253.29; P < 0.001). CONCLUSIONS: Pregnancy and blood transfused had high allo-immunization rate for class I HLA antigens. While re-transplantation patients had high allo-immunization rate for both the HLA classes (HLA- class I and HLA- class II). Re-transplantation group showed significantly stronger antibody strength, followed by pregnancy and then transfusion.