Mouse IgG2b Research Articles

Combining Cellular Immunization and Phage Display Screening Results in Novel, FcγRI-Specific Antibodies.

Antibodies that specifically bind to individual human fragment crystallizable γ receptors (FcγRs) are of interest as research tools in studying immune cell functions, as well as components in bispecific antibodies for immune cell engagement in cancer therapy. Monoclonal antibodies for human low-affinity FcγRs have been successfully generated by hybridoma technology and are widely used in pre-clinical research. However, the generation of monoclonal antibodies by hybridoma technology that specifically bind to the high-affinity receptor FcγRI is challenging. Monomeric mouse IgG2a, IgG2b, and IgG3 bind human FcγRI with high affinity via the Fc part, leading to an Fc-mediated rather than a fragment for antigen binding (Fab)-mediated selection of monoclonal antibodies. Blocking the Fc-binding site of FcγRI with an excess of human IgG or Fc during screening decreases the risk of Fc-mediated interactions but can also block the potential epitopes of new antibody candidates. Therefore, we replaced hybridoma technology with phage display of a single-chain fragment variable (scFv) antibody library that was generated from mice immunized with FcγRI-positive cells and screened it with a cellular panning approach assisted by next-generation sequencing (NGS). Seven new FcγRI-specific antibody sequences were selected with this methodology, which were produced as Fc-silent antibodies showing FcγRI-restricted specificity.

Treatment with doxorubicin and PD-1/CTLA-4 blockade improves T cell activation and anti-tumor efficacy in MCA-205 murine fibrosarcoma

Abstract Immune checkpoint inhibitors (ICIs) against PD-1 and CTLA-4 produce responses in about 20% of adult soft tissue sarcomas (STSs), however, most patients do not respond, potentially from poor T cell activation. Standard chemotherapy for STSs includes the anthracycline doxorubicin (DOX) which has been shown in preclinical cancer models to induce immunogenic cell death (ICD) via tumor production of type I interferons (IFN). We hypothesized that induction of ICD and IFN release by doxorubicin would enhance T cell activation, infiltration, and boost anti-tumor efficacy when combined with dual anti-PD-1/CTLA-4 blockade in the MCA-205 murine fibrosarcoma model. MCA-205 tumors were generated in C57BL/6 mice and were treated with DOX, anti-PD1 (RMP1–14), anti-CTLA4 (9D9 mouse IgG2b) or a mouse surrogate of botensilimab, an anti-CTLA4 (9D9 mouse IgG2b.DLE) antibody with enhanced binding to FcγR, or combination DOX plus anti-PD-1/CTLA-4. We found reduction of tumor growth and improved survival (median survival not reached) with DOX/PD-1/CTLA-4 compared to DOX (median survival 29.5 days). This correlated with a 2-fold increased influx of TILs as measured by flow cytometry and immunohistochemistry, with a marked increase in CD8:CD4 T cell ratio with increased expression of activation markers (PD-1, 4–1BB and TIM3). TCR Vβ clonotyping revealed a shift in clonotype frequency with DOX/PD-1/CTLA-4 relative to other treatment groups. Pathway analysis from bulk RNA sequencing demonstrated upregulation of T cell activation in DOX/PD-1/CTLA-4 tumors compared to control tumors. Further work will correlate these findings with human STS patients receiving DOX plus CTLA-4/PD-1 blockade in an ongoing clinical trial (NCT04028063). This work was supported by grants from the University of Colorado Cancer Center (P30CA046934) and the University of Colorado School of Medicine.

Development of highly specific peptibody against Porcine epidemic diarrhea virus

Abstract Porcine epidemic diarrhea virus (PEDV) is an etiological agent of porcine epidemic diarrhea (PED), causing dehydration, vomiting, and acute watery diarrhea. Because it has a high mortality rate in neonatal piglets, PEDV is one of the critical pathogens affecting the swine industry. Since PEDV has shown rapid spreading in herds of swine, early diagnosis of this virus is very important to control the disease. Serological diagnosis test is a simple, rapid method for detecting PEDV infected pigs, and the development of PEDV-specific antibodies is essential for an accurate serological diagnostic test. Because conventional methods of development of monoclonal antibodies are time and hand-consuming work, recombinant antibodies are considered as an alternative strategy for the development of target-specific monoclonal antibodies. Peptibody is one of the promising recombinant antibodies that target-specific peptide is fused with the Fc domain of immunoglobulin G. The peptibody which have a specificity of peptides and immune function of Fc region as advantages have developed for therapeutic and diagnostic purpose. Here, we generated a PEDV-specific peptibody (PEDV-PB) that is fused with PEDV-specific peptide and mouse IgG2b Fc region. PEDV-specific peptide sequences were discovered by random phage display library and PEDV-PB was produced from Chinese hamster ovary (CHO) cells. PEDV-PB showed highly specific binding in several serological tests for PEDV, such as western blotting, immunofluorescence assay, and enzyme-linked immunosorbent assay. Our results suggest that PEDV-PB is very useful for the development of a rapid detection kit for PEDV.

TrMab-6 Exerts Antitumor Activity in Mouse Xenograft Models of Breast Cancers

Trophoblast cell surface antigen 2 (TROP2) has been reported to be overexpressed in many cancers, and is involved in cancer cell proliferation, invasion, and metastasis. We previously developed a highly sensitive anti-TROP2 monoclonal antibody (mAb) (clone TrMab-6; mouse IgG2b, kappa) using a Cell-Based Immunization and Screening method. TrMab-6 is useful for investigations using flow cytometry, Western blotting, and immunohistochemistry and possesses antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against TROP2-expressing triple-negative breast cancer (TNBC) cell lines, such as MDA-MB-231 and MDA-MB-468. This study investigated whether TrMab-6 possesses in vivo antitumor activities via ADCC/CDC activities using mouse xenograft models of TNBC cell lines. In vivo experiments on MDA-MB-231 and MDA-MB-468 xenografts revealed that TrMab-6 significantly reduced tumor growth compared with normal mouse IgG treatment. The findings of this study suggest that TrMab-6 is a promising treatment option for TROP2-expressing TNBC.

Epitope Mapping of an Anti-CD20 Monoclonal Antibody (C20Mab-60) Using the HisMAP Method.

CD20 is one of the B-lymphocyte antigens and an effective target for the detection and treatment of B cell lymphomas; specific and sensitive monoclonal antibodies (mAbs) are required thus for their diagnosis. Recently, we developed a novel anti-CD20 mAb (clone C20Mab-60), which is not only useful for flow cytometry but also for Western blot and immunohistochemical analyses. However, the epitope of C20Mab-60 has not been determined. To clarify the binding region of mAbs against their target molecules, it is essential to understand the pharmacological function of each mAb. In this study, we aimed to identify the epitope of C20Mab-60 for CD20 using the novel histidine tag (His-tag) insertion for epitope mapping (HisMAP) method. We first established an anti-His-tag mAb, HisMab-1 (mouse IgG2b, kappa), by immunizing mice with recombinant proteins containing an N-terminal His-tag. Although HisMab-1 detected the 4x, 5x, and 6xHis tag-inserted CD20 proteins using flow cytometry, 5xHis tag was selected. While HisMab-1 recognized all the 5xHis tag-inserted CD20 from the 142nd to the 183rd amino acid (aa), C20Mab-60 did not react with the 5xHis tag-inserted CD20 from the 171st to the 174th aa. These results indicate that the main epitope of C20Mab-60 for CD20 is a peptide from 171st to 174th aa of CD20. HisMAP method could be advantageous in the determination of the critical epitope of functional mAbs against many target molecules.

An anti‑TROP2 monoclonal antibody TrMab‑6 exerts antitumor activity in breast cancer mouse xenograft models.

Trophoblast cell surface antigen 2 (TROP2), reported to be overexpressed in several types of cancer, is involved in cell proliferation, invasion, metastasis, and poor prognosis of many types of cancer. Previously, a highly sensitive anti-TROP2 monoclonal antibody (clone TrMab-6; mouse IgG2b, κ) was developed using a Cell-Based Immunization and Screening (CBIS) method. TrMab-6 was useful for investigations using flow cytometry, western blot, and immunohistochemistry. The aim of the present study was to investigate whether TrMab-6 possesses in vitro antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) activities or in vivo antitumor activities using mouse xenograft models of TROP2-overexpressed CHO-K1 (CHO/TROP2) and breast cancer cell lines, including MCF7, MDA-MB-231, and MDA-MB-468. In vitro experiments revealed that TrMab-6 strongly induced ADCC and CDC activities against CHO/TROP2 and the three breast cancer cell lines, whereas it did not show those activities against parental CHO-K1 and MCF7/TROP2-knockout cells. Furthermore, in vivo experiments on CHO/TROP2 and MCF7 ×enografts revealed that TrMab-6 significantly reduced tumor growth, whereas it did not show antitumor activities against parental CHO-K1 and MCF7/TROP2-knockout xenografts. The findings suggest that TrMab-6 is a promising treatment option for TROP2-expressing breast cancers.

Development and characterization of TrMab‑6, a novel anti‑TROP2 monoclonal antibody for antigen detection in breast cancer.

Trophoblast cell-surface antigen 2 (TROP2) is a type I transmembrane glycoprotein that is overexpressed in a number of cancer types, including triple-negative breast cancer. The current study aimed to develop a highly sensitive and specific monoclonal antibody (mAb) targeting TROP2, which could be used to evaluate TROP2 expression using flow cytometry, western blot analysis and immunohistochemistry by employing the Cell-Based Immunization and Screening (CBIS) method. The established anti-TROP2 mAb, TrMab-6 (mouse IgG2b, κ), detected TROP2 on PA-tagged TROP2-overexpressing Chinese hamster ovary-K1 (CHO/TROP2-PA) and breast cancer cell lines, including MCF7 and BT-474 using flow cytometry. Western blot analysis indicated a 40 kDa band in lysates prepared from CHO/TROP2-PA, MCF7 and BT-474 cells. Furthermore, TROP2 in 57/61 (93.4%) of the breast cancer specimens was strongly detected using immunohistochemical analysis with TrMab-6. In conclusion, the current study demonstrated that TrMab-6 may be a valuable tool for the detection of TROP2 in a wide variety of breast cancer types.

Epitope Mapping of the Anti-Diacylglycerol Kinase Monoclonal Antibody DhMab-4 for Immunohistochemical Analysis

Diacylglycerol kinase (DGK) plays a pivotal role in intracellular signaling pathways in mammals. Activated G protein-coupled receptor activates phospholipase C (PLC) through heterotrimeric G protein, following which PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3). DGK catalyzes DG phosphorylation to produce phosphatidic acid. DG and phosphatidic acid function as second messengers and their intracellular concentrations are regulated by DGK; therefore, DGK plays an important role in regulating many biological processes. There are ten DGK isozymes, of which DGKη is classified as a type II DGK. Reports have shown that DGKη is associated with several diseases; for example, it is highly expressed in the hippocampus and cerebellum and is a key element in bipolar disorder. Although a DGKη-specific monoclonal antibody (mAb) is necessary to reveal the association between the expression of DGKη and diseases, an anti-DGKη mAb for immunohistochemistry has not yet been established. In this study, we established a specific anti-human DGKη (hDGKη) mAb, DhMab-4 (mouse IgG2b, kappa). DhMab-4 strongly stained Purkinje cells of human cerebellum in immunohistochemistry analysis. For epitope mapping of DhMab-4, we produced deletion or point mutants of hDGKη and performed western blotting to determine the binding epitope of DhMab-4. DhMab-4 reacted with dN745 mutant but not with dN750 mutant, indicating that the N-terminus of the DhMab-4 epitope is located between amino acids 745 and 750. More detailed analysis using point mutants demonstrated that five mutants, that is, D747A, P748A, F749A, G750A, and T752A, were not detected by DhMab-4. These results indicate that Asp747, Pro748, Phe749, Gly750, and Thr752 are important for DhMab-4 binding to hDGKη.

Corrigendum.

ImmunologyVolume 159, Issue 2 p. 242-242 CorrigendumFree Access Corrigendum This article corrects the following: CD6 monoclonal antibodies differ in epitope, kinetics and mechanism of action Lee I. Garner, Andrew Hartland, Johannes Breuning, Marion H. Brown, Volume 155Issue 2Immunology pages: 273-282 First Published online: June 13, 2018 First published: 14 November 2019 https://doi.org/10.1111/imm.13113AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat The article1 should be read with reference to new data2 for the correct specificity of the CD6 mAbs, OX124, OX125 and OX126.3, 4 Domain specificity for OX124, OX125 and OX126 should be deleted and the name of the antibody given. As the focus of the article is domain 1 mAbs, the overall conclusions are not altered. Specific alterations to the text are: Summary, p273. CD6 domain 1, OX125 and OX126 mAbs were equally effective in triggering interleukin-2 production…….CD6 domain 1 mAbs hindered binding of multivalent immobilised CD166 but were inferior compared with blocking by soluble CD166 or another CD6 mAb. Introduction, p274. Immunisation with soluble recombinant CD6 led to the production of novel CD6 mAbs(6). Materials and Methods, Monoclonal antibodies, p274. CD6, OX124 (mouse IgG1), OX125 (mouse IgG2b) and OX126 (mouse IgG1)(6) Materials and Methods, Flow cytometry, p275. CD6 mAb (OX126) Results, p277 and Figure 3. CD6 domain 1, OX125 and OX126 mAbs….. Results, p278. …CD6 mAbs, OX124, OX125 and OX126 specific for different epitopes on CD6 for efficacy …a CD6 mAb, OX125… interfere with OX126 binding to CD6. We began by asking do CD6 domain 1 mAbs hinder binding of OX126(6)……interactions of OX126. Results, p279 and Figure 4. CD6 mAb, OX126. Results, p279 and Figure 5. The data (Figure 5) are inconsistent with the specificity of OX1262 and indicate an antibody labelling error.3, 4 Read: CD6 domain 1 mAbs are less effective compared with another CD6 mAb….another CD6 mAb which was superior….another CD6 mAb was more effective >UMCD6>itolizumab at Discussion, p280. binding to domain 1 and by OX125 or OX126 in the efficacy of triggering.. Discussion, p281…competition experiment with a CD6 mAb, OX126..Delete text: “As none …soluble CD166(6)”. UMCD6 is not as efficacious at blocking CD6/CD166 interactions compared with another CD6 mAb References 1Garner LI, Hartland A, Breuning J, Brown MH. CD6 monoclonal antibodies differ in epitope, kinetics and mechanism of action. Immunology 2018; 155: 273– 82. 2Santos RF, Oliveira L, Brown MH, Carmo AM. Domain-specific CD6 monoclonal antibodies identify CD6 isoforms generated by alternative-splicing. Immunology 2019; 155: 273– 82. 3Hassan NJ, Simmonds SJ, Clarkson NG, Hanrahan S, Puklavec MJ, Bomb M et al. Second correction for Hassan et al., CD6 regulates T-cell responses through activation-dependent recruitment of the positive regulator SLP-76. Mol Cell Biol 2019; 39: e00054- 19. 4Hassan NJ, Simmonds SJ, Clarkson NG, Hanrahan S, Puklavec MJ, Bomb M et al. CD6 regulates T-cell responses through activation-dependent recruitment of the positive regulator SLP-76. Mol Cell Biol 2006; 26: 6727– 38. Volume159, Issue2February 2020Pages 242-242 ReferencesRelatedInformation

Abstract 4588: Characterization of anti-CTLA-4 effects on tumor growth and immune microenvironment in a syngeneic CT26.WT colon carcinoma mouse model

Abstract Colorectal cancer is the third most common type of cancer and one of the leading causes of cancer related deaths. It is a group of various diseases at molecular level, which can make targeted drug development challenging. However, novel therapeutics including immunotherapies have shown their potential to be utilized in the future also when treating colorectal cancer. One promising example is ipilimumab, a monoclonal antibody targeting CTLA-4 (cytotoxic T lymphocyte antigen-4, also known as CD152). CTLA-4 is a cell surface receptor expressed on activated T and B lymphocytes. It regulates immunological tolerance and downregulates immune responses. The aim of the study was to characterize the effects of anti-CTLA-4 on tumor growth, survival and tumor immune microenvironment of mice subcutaneously inoculated with mouse colon carcinoma CT26.WT cells. CT26.WT cells were subcutaneously inoculated into female Balb/c mice (n=27). Body weights and tumor dimensions were determined two times a week during the course of the study. The mice were randomized to three groups (n=9/group) with similar tumor volume and variation and dosing was started when an average tumor size of approximately 50 mm³ was reached (day 7). Anti-CTLA-4 antibody (9D9, 10 mg/kg), an isotype control (mouse IgG2b) and vehicle (PBS) were administered (i.p.) twice a week. Tumor growth and survival were followed for 26 days after inoculation of the cancer cells. The mice were sacrificed individually when the maximum tumor volume (1500 mm³) was reached, the tumors were ulcerated or at the latest on study day 26. Tumor samples were collected for histological and immunohistochemical analysis and stained for helper T cells (CD4), cytotoxic T cells (CD8), myeloid-derived suppressor cells (MDSC; CD11b), tumor-associated macrophages (F4/80), and inflammatory monocytes and neutrophils (FCGR1/CD64) for characterization of tumor immune microenvironment. Maximum tumor volume and tumor ulceration were observed the earliest on study day 17. Anti-CTLA-4 suppressed tumor growth, and all mice in the group receiving anti-CTLA-4 were still in the study on day 26. Only one and four mice were left in the vehicle and isotype control groups, respectively. Immunohistochemical staining suggested an increase of CD4+ tumor-infiltrating lymphocytes (TILs) in anti-CTLA-4 treated mice. Furthermore, more TAMs were observed in tumors of anti-CTLA-4 treated mice, especially in the borderlines of necrotic areas. Large quantity of CD11b+ MDSCs and inflammatory cells were observed in CT26.WT tumors. CT26.WT subcutaneous model can be utilized for studying the effects of immunotherapies, and anti-CTLA-4 treatment suppressed tumor growth in this model. CT26.WT tumors represent a highly immunogenic hot tumor type with high immune cell infiltration, associated with a good efficacy of anti-CTLA-4 treatment in the model. Citation Format: Jenni H. Mäki-Jouppila, Tiina E. Kähkönen, Mari I. Suominen, Jussi M. Halleen, Jenni Bernoulli. Characterization of anti-CTLA-4 effects on tumor growth and immune microenvironment in a syngeneic CT26.WT colon carcinoma mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4588.

Roles of Fc Domain and Exudation in L2 Antibody-Mediated Protection against Human Papillomavirus.

To address how L2-specific antibodies prevent human papillomavirus (HPV) infection of the genital tract, we generated neutralizing monoclonal antibodies (MAbs) WW1, a rat IgG2a that binds L2 residues 17 to 36 (like mouse MAb RG1), and JWW3, a mouse IgG2b derivative of Mab24 specific for L2 residues 58 to 64. By Western blotting, WW1 recognized L2 of 29/34 HPV genotypes tested, compared to only 13/34 for RG1 and 25/34 for JWW3. WW1 IgG and F(ab')2 bound HPV16 pseudovirions similarly; however, whole IgG provided better protection against HPV vaginal challenge. Passive transfer of WW1 IgG was similarly protective in wild-type and neonatal Fc receptor (FcRn)-deficient mice, suggesting that protection by WW1 IgG is not mediated by FcRn-dependent transcytosis. Rather, local epithelial disruption, required for genital infection and induced by either brushing or nonoxynol-9 treatment, released serum IgG in the genital tract, suggesting Fc-independent exudation. Depletion of neutrophils and macrophages reduced protection of mice upon passive transfer of whole WW1 or JWW3 IgGs. Similarly, IgG-mediated protection by L2 MAbs WW1, JWW3, and RG1 was reduced in Fc receptor knockout compared to wild-type mice. However, levels of in vitro neutralization by WW1 IgG were similar in TRIM21 knockout and wild-type cells, indicating that Fc does not contribute to antibody-dependent intracellular neutralization (ADIN). In conclusion, the Fc domain of L2-specific IgGs is not active for ADIN, but it opsonizes bound extracellular pseudovirions for phagocytes in protecting mice from intravaginal HPV challenge. Systemically administered neutralizing IgG can access the site of infection in an abrasion via exudation without the need for FcRn-mediated transcytosis.IMPORTANCE At least 15 alpha HPV types are causative agents for 5% of all cancers worldwide, and beta types have been implicated in nonmelanoma skin cancer, whereas others produce benign papillomas, such as genital warts, associated with considerable morbidity and health systems costs. Vaccines targeting the minor capsid protein L2 have the potential to provide broad-spectrum immunity against medically relevant HPVs of divergent genera via the induction of broadly cross-neutralizing serum IgG. Here we examine the mechanisms by which L2-specific serum IgG reaches the viral inoculum in the genital tract to effect protection. Abrasion of the vaginal epithelium allows the virus to access and infect basal keratinocytes, and our findings suggest that this also permits the local exudation of neutralizing IgG and vaccine-induced sterilizing immunity. We also demonstrate the importance of Fc-mediated phagocytosis of L2 antibody-virion complexes for humoral immunity, a protective mechanism that is not detected by current in vitro neutralization assays.

Abstract 953: LRRC15 is a novel antigen in sarcoma and the therapeutic target of the antibody-drug conjugate (ADC) ABBV-085

Abstract Background: Leucine rich repeat containing 15 (LRRC15) is a TGFβ-regulated structural protein that is highly expressed on cancer-associated fibroblasts (CAFs) in the stromal microenvironment of many solid tumors, as well as directly on cancer cells of mesenchymal origin. Soft tissue sarcomas (STS) and bone sarcomas (BS) represent a diverse family of mesenchymal malignancies that can develop at any anatomic site and that comprise more than 70 histopathologic subtypes. After screening LRRC15 expression across a variety of sarcoma histologies, we evaluated the antitumor activity of ABBV-085, an MMAE (monomethyl auristatin E) containing antibody-drug conjugate directed against LRRC15 (mouse, cyno, human), in patient-derived xenograft (PDX) models of selected sarcomas with varying levels of LRRC15 expression. Methods: LRRC15 expression/intensity in sarcoma histologies was performed by immunohistochemical (IHC) staining (Leica Bond RX, Bond Polymer Refine Kit) with a human LRRC15 specific mouse IgG2b antibody. Based on LRRC15 expression levels, STS and BS tumor fragments (PDXs) were implanted into NSG mice. Mice with growing tumors were then selected to evaluate the in vivo efficacy of ABBV-085 monotherapy (6 mg/kg). Results: LRRC15 expression was evaluated in 340 human sarcoma tumors representing a variety of STS and BS histologic subtypes. LRRC15 IHC expression was determined by scoring the percentage of positive cells, together with the intensity of staining (score of 0 for negative, 1 for weak, 2 for moderate, and 3 for strong staining), for the cancer cells and stroma, respectively. A stringent cut-off for strong LRRC15 positivity was defined as ≥2+ intensity in ≥50% of the cancer or stromal area. Strong LRRC15 expression was observed in several sarcoma subsets: 67% (14/21) of osteosarcoma (OS) tumor samples, 64% (23/36) of undifferentiated pleomorphic sarcoma (UPS), 18% (8/44) of leiomyosarcomas (LMS) and 17% (6/35) of liposarcomas (LPS). Significant antitumor activity including regressions and cures was induced by ABBV-085 in LRRC15-positive STS and BM PDX models, when compared with isotype-control treated mice. The ABBV-085-induced efficacy in osteosarcoma PDX models was superior to current standard-of-care therapies when dosed maximally in mice. In addition, ABBV-085 demonstrated efficacy in different LRRC15 positive STS subtypes including UPS, LMS and LPS. ABBV-085 was well tolerated with minimal to no body weight loss observed. Conclusions: LRRC15 is highly expressed in the majority of human osteosarcomas and undifferentiated pleomorphic sarcomas, as well as in a range of other STS and BS subtypes. ABBV-085 demonstrates promising preclinical antitumor efficacy in LRRC15-positive PDX models of STS and BS. ABBV-085 is currently being investigated in an ongoing phase 1 study in soft tissue sarcomas (including undifferentiated pleomorphic sarcoma) and osteosarcoma. Citation Format: Eytan Ben-Ami, Ying Huang, Prafulla C. Gokhale, Benjamin Eschle, Lisa Durkin, Jonathan Hickson, Mien Sho, Susan Morgan-Lappe, Kurt Gish, Dominic W. Lai, Randy R. Robinson, Diane Hollenbaugh, Eric D. Hsi, Debra T. Chao, George D. Demetri, James W. Purcell. LRRC15 is a novel antigen in sarcoma and the therapeutic target of the antibody-drug conjugate (ADC) ABBV-085 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 953.

Mouse IgG2c Fc loop residues promote greater receptor-binding affinity than mouse IgG2b or human IgG1.

The structures of non-human antibodies are largely unstudied despite the potential for the identification of alternative structural motifs and physical properties that will benefit a basic understanding of protein and immune system evolution as well as highlight unexplored motifs to improve therapeutic monoclonal antibody. Here we probe the structure and receptor-binding properties of the mouse IgG2c crystallizable fragment (Fc) to compare to mouse IgG2b and human IgG1 Fcs. Models of mIgG2c Fc determined by x-ray crystallography with a complex-type biantennary (to 2.05 Å) or a truncated (1)GlcNAc asparagine-linked (N)-glycan attached (to 2.04 Å) show differences in key regions related to mouse Fc γ receptor IV (mFcγRIV) binding. Mouse IgG2c forms different non-bonded interactions between the BC, DE and FG loops than the highly-conserved mIgG2b and binds to FcγRIV with 4.7-fold greater affinity in the complex-type glycoform. Secondary structural elements surrounding the Asn297 site of glycosylation form longer beta strands in the truncated mIgG2c Fc glycoform when compared to mIgG2c with the complex-type N-glycan. Solution NMR spectroscopy of the N-linked (1)GlcNAc residues show differences between mIgG2b, 2c and hIgG1 Fc that correlate to receptor binding affinity. Mutations targeting differences in mIgG2 DE and FG loops decreased affinity of mIgG2c for FcγRIV and increased affinity of mIgG2b. Changes in NMR spectra of the mutant Fc proteins mirrored these changes in affinity. Our studies identified structural and functional differences in highly conserved molecules that were not predicted from primary sequence comparison.

Efficient Generation of Bispecific Murine Antibodies for Pre-Clinical Investigations in Syngeneic Rodent Models

Therapeutic concepts exploiting tumor-specific antibodies are often established in pre-clinical xenograft models using immuno-deficient mice. More complex therapeutic paradigms, however, warrant the use of immuno-competent mice, that more accurately capture the relevant biology that is being exploited. These models require the use of (surrogate) mouse or rat antibodies to enable optimal interactions with murine effector molecules. Immunogenicity is furthermore decreased, allowing longer-term treatment. We recently described controlled Fab-arm exchange (cFAE) as an easy-to-use method for the generation of therapeutic human IgG1 bispecific antibodies (bsAb). To facilitate the investigation of dual-targeting concepts in immuno-competent mice, we now applied and optimized our method for the generation of murine bsAbs. We show that the optimized combinations of matched point-mutations enabled efficient generation of murine bsAbs for all subclasses studied (mouse IgG1, IgG2a and IgG2b; rat IgG1, IgG2a, IgG2b, and IgG2c). The mutations did not adversely affect the inherent effector functions or pharmacokinetic properties of the corresponding subclasses. Thus, cFAE can be used to efficiently generate (surrogate) mouse or rat bsAbs for pre-clinical evaluation in immuno-competent rodents.

Amelioration of Immune Thrombocytopenia in Human CD44 Transgenic Mice By Anti-Human CD44 Antibodies

Selected monoclonal antibodies to murine CD44 are able to successfully ameliorate murine immune thrombocytopenia (ITP) and other inflammatory diseases at a 3 log fold lower dose than IVIg. Since not all murine CD44 antibodies were successful and, like IVIg, the anti-inflammatory mechanism involved is speculative, progress toward human antibodies has been stymied. To potentially develop a therapeutic antibody for patients, we generated transgenic mice expressing the extracellular and transmembrane sequences of human CD44 linked to the murine intracellular CD44 sequence (huCD44) with knockout of murine CD44. Flow cytometric analysis revealed that these mice express huCD44 on leukocytes, but not on platelets or red blood cells.HuCD44 mice injected with the anti-platelet antibody MWReg30 develop significant thrombocytopenia comparable to normal mice. Utilizing the huCD44 mouse model, we tested 5 monoclonal antibodies reactive with human CD44 of different isotypes; mouse IgG1, mouse IgG2a, mouse IgG2b, rat IgG2a, and rat IgG2b. We report here that pretreatment of mice with all of these antibodies, with the exception of the mouse IgG2b clone all ameliorated thrombocytopenia to the same extent as IVIg, but given at a 3 log-fold lower dosage than IVIg. Interestingly, the mouse IgG2b antibody that was not successful bound to white blood cells as well as the successful antibodies. When mice were re-injected with anti-platelet antibody 24 hr post anti-huCD44 antibody injection, they were still protected from thrombocytopenia at 48 hr, suggesting that these antibodies exhibit durable protection.The clinical effectiveness of these antibodies does not appear to be related to their extent of huCD44 binding or IgG subtype. In addition, the effectiveness of these antibodies may not be restricted or related to specific activating IgG Fc receptors (FcγR) as the successful mouse IgG1 subtype is only known to interact with FcγRIII, while the IgG2b subtype can bind activating FcγRI, III and IV with only one-of-two of this subtype having therapeutic activity.In conclusion we have developed, to our knowledge, the first huCD44 transgenic mouse and demonstrate herein that selected antibodies to huCD44 can ameliorate immune thrombocytopenia. These data suggest that some anti-CD44 antibodies may offer a new therapeutic modality for patients with ITP. DisclosuresNo relevant conflicts of interest to declare.

LpMab-19 Recognizes SialylatedO-Glycan on Thr76 of Human Podoplanin

Human podoplanin (hPDPN) is expressed in lymphatic vessels, pulmonary type-I alveolar cells, and renal glomerulus. The hPDPN/C-type lectin-like receptor-2 (CLEC-2) interaction is involved in platelet aggregation and cancer metastasis. High expression of hPDPN in cancer cells or cancer-associated fibroblasts (CAFs) leads to a poor prognosis for cancer patients. In our previous research, we reported on several anti-hPDPN monoclonal antibodies (mAbs), including LpMab-2, LpMab-3, LpMab-7, LpMab-9, LpMab-12, LpMab-13, and LpMab-17 of mouse IgG1 subclass, which were produced using CasMab technology. Here we produced a novel anti-hPDPN mAb LpMab-19 of mouse IgG2b subclass. Flow cytometry revealed that the epitope of LpMab-19 includes O-glycan, which is attached to Thr76 of hPDPN. We further identified the minimum epitope of LpMab-19 as Thr76-Arg79 of hPDPN. Immunohistochemistry revealed that LpMab-19 is useful for detecting not only normal cells, including lymphatic vessels, but also glioblastoma and oral squamous cell carcinoma cells. LpMab-19 could be useful for investigating the physiological function of O-glycosylated hPDPN.

IgG subclasses determine pathways of anaphylaxis in mice

Animal models have demonstrated that allergen-specific IgG confers sensitivity to systemic anaphylaxis that relies on IgG Fc receptors (FcγRs). Mouse IgG2a and IgG2b bind activating FcγRI, FcγRIII, and FcγRIV and inhibitory FcγRIIB; mouse IgG1 binds only FcγRIII and FcγRIIB. Although these interactions are of strikingly different affinities, these 3 IgG subclasses have been shown to enable induction of systemic anaphylaxis. We sought to determine which pathways control the induction of IgG1-, IgG2a-, and IgG2b-dependentpassive systemic anaphylaxis. Mice were sensitized with IgG1, IgG2a, or IgG2b anti-trinitrophenyl mAbs and challenged with trinitrophenyl-BSA intravenously to induce systemic anaphylaxis that was monitored by using rectal temperature. Anaphylaxis was evaluated in mice deficient for FcγRs injected with mediator antagonists or in which basophils, monocytes/macrophages, or neutrophils had been depleted. FcγR expression was evaluated on these cells before and after anaphylaxis. Activating FcγRIII is the receptor primarily responsible for all 3 models of anaphylaxis, and subsequent downregulation of this receptor was observed. These models differentially relied on histamine release and the contribution of mast cells, basophils, macrophages, and neutrophils. Strikingly, basophil contribution and histamine predominance in mice with IgG1- and IgG2b-induced anaphylaxis correlated with the ability of inhibitory FcγRIIB to negatively regulate these models of anaphylaxis. We propose that the differential expression of inhibitory FcγRIIB on myeloid cells and its differential binding of IgG subclasses controls the contributions of mast cells, basophils, neutrophils, and macrophages to IgG subclass-dependent anaphylaxis. Collectively, our results unravel novel complexities in the involvement and regulation of cell populations in IgG-dependent reactions invivo.

Treatment with Anti-HMGB1 Monoclonal Antibody Does Not Affect Lupus Nephritis in MRL/lpr Mice.

High mobility group box 1 (HMGB1) is a nuclear DNA binding protein that acts as an alarmin when secreted. HMGB1 is increased in systemic lupus erythematosus and might represent a potential therapeutic target. We investigated whether treatment with an anti-HMGB1 antibody affects the development of lupus nephritis in MRL/lpr mice. Seven-week-old MRL/lpr mice were injected intraperitoneally twice weekly for 10 wks with 50μg monoclonal anti-HMGB1 (2G7, mouse IgG2b) (n = 12) or control antibody (n = 11). Control MRL/MPJ mice (n = 10) were left untreated. Every 2 wks, blood was drawn and urine was collected at wk 7, 11 and 17. Mice were sacrificed at 17 wks for complete disease evaluation. Plasma HMGB1 and anti-HMGB1 levels were increased in MRL/lpr mice compared with control MRL/MPJ mice. There were no differences in albuminuria, urine HMGB1 and plasma levels of complement C3, anti-dsDNA and proinflammatory cytokines between untreated and treated mice at any time point. Lupus nephritis of mice treated with anti-HMGB1 monoclonal antibody (mAb) was classified as class III (n = 3) and class IV (n = 9), while mice treated with control mAb were classified as class II (n = 4), class III (n = 2) and class IV (n = 5). IgG and C3 deposits in kidneys were similar in mice treated with anti-HMGB1 mAb or control mAb. In conclusion, treatment with monoclonal anti-HMGB-1 antibody 2G7 does not affect development of lupus nephritis, disease progression or proinflammatory cytokine levels in MRL/lpr mice. This result indicates that blocking of HMGB1 by this neutralizing antibody does not affect lupus nephritis in MRL/lpr mice.

Use of Monoclonal Antibodies to RBC Antigens As a Therapeutic to Allow Incompatible Transfusion

Background: Transfusion of RBCs is an essential therapy to treat myriad diseases. In excess of 340 different RBC alloantigens have now been described; thus, every non-autologous unit of RBCs constitutes a foreign alloantigen. Once a patient makes an alloantibody, in many cases, they can no longer receive RBCs expressing the recognized alloantigen. For chronically transfused patients, who make multiple alloantibodies, identifying sufficient compatible RBC units can become difficult, leading to morbidity, and in some cases, mortality. Thus alloimmunization is a significant clinical problem. As the vast majority of RBC alloantigens are single amino acid polymorphisms, they presumably constitute small epitopes. We hypothesized that monoclonal antibodies against human blood group antigens, engineered so as to lack effector function, would bind to the offending alloantigen in a fashion that would both block binding of recipient alloantibody and allow normal RBC circulation.Methods: Wild-type mice were alloimmunized with transgenic RBCs expressing Kell (K1), splenocytes were fused with myeloma lines, and monoclonal antibodies were isolated. An antibody with specificity for the K1 antigen was isolated (PUMA1). The cDNA for both heavy and light chains were cloned and the variable regions were ligated upstream of constant regions of mouse IgG1, IgG2a, IgG2b, and IgG3. Each anti-K1 IgG subtype was expressed by transfecting constructs into COS cells and antibodies were purified by protein A/G chromatography. The purified antibodies were passively administered to wild type mice, which were then transfused with K1 RBCs. RBC survivals were monitored by flow cytometry. PUMA1 was also humanized with both naturally occurring human IgG constant regions and also a mutant IgG lacking binding sites for complement and Fc-gamma-receptors.Results: Each of the IgGs cleared K1 transgenic RBCs (but not wild-type RBCs); however, clearance potency varied with IgG subtype, with a rank order of IgG2a>IgG1>IgG2b>IgG3. To test the ability of the less hemolytic forms to block clearance by the more hemolytic forms, wild-type mice were infused with PUMA1 IgG2a. K1 RBCs were pre-incubated with PBS, 10ug IgG2b or 10ug IgG3 and then transfused. Alternatively, PBS, 10ug IgG2b or 10ug IgG3 was infused into the recipient mice, right before transfusion of K1 RBCs. Both approaches yielded similar results. Experimental animals that received IgG2a followed by IgG2b or IgG3 (either incubation or infusion) had a 24hr K1 RBC recovery ranging from 72-82%, while mice that received IgG2a followed by PBS had a 41-44% recovery. The humanized forms have been expressed and maintain specific binding to the K1 alloantigen.Conclusions: Together, the data presented herein demonstrate the ability of less hemolytic forms of anti-K1 alloantibodies to serve as a therapeutic to decrease delayed hemolytic transfusion reactions by more hemolytic forms, in a murine model using murine IgG forms of PUMA1. Ongoing in vivo murine studies are assessing the humanized and mutated forms of PUMA1 to serve as a therapeutic reagent that can block access of hemolytic anti-K1 alloantibodies to incompatible RBCs, both in wild-type mice and also in mice with humanized Fc-gamma receptors. The ultimate goal of these studies is the development of therapeutic modified antibodies that can allow safe and effective incompatible transfusion in patients alloimmunized to RBC antigens. For a multiply alloimmunized patient, being able to block one or two antibodies would allow identification of units that were negative for other antibody specificities; thus, therapeutics of this type against a limited number of common alloantigens would have substantial impact. DisclosuresZimring:BloodworksNW: Patents & Royalties: Patent Application filed on technology in this abstract - no royalties; Immucor Inc.: Research Funding.

Production and Purification of a Polyclonal Antibody Against Purified Mouse IgG2b in Rabbits Towards Designing Mouse Monoclonal Isotyping Kits.

Mouse IgG subclasses containing IgG1, IgG2a, IgG2b and IgG3 have been defined and described both physiochemically and immunologically. Sepharose beads conjugated with protein A affinity chromatography was used for purification of mouse IgG2b. Sodium citrate buffer (0.1 M, pH: 3.5) was used for separation of mouse IgG2b. Verification of the purified fractions was monitored by SDS-PAGE (polyacrylamide gel electrophoresis) in reducing condition. Immunized rabbit serum was collected and precipitated at the final concentration of 50% ammonium sulfate. After dialysis against tris-phosphate buffer (pH: 8.1) ion exchange chromatography column was used for purification of rabbit anti-mouse IgG2b. The periodate method was performed for conjugation with some variations. After conjugation, direct ELISA was used to determine the titer of HRP conjugated rabbit IgG against mouse IgG2b. The titer of rabbit anti-mouse IgG2b that determined by ELISA was 32000. The purity of rabbit anti-mouse IgG2b was about 95%. The optimum dilution of prepared HRP conjugated IgG was 1:10000. This study showed that ion-exchange chromatography and affinity chromatography could be appropriate techniques for purification of mouse IgG and IgG subclasses respectively. This study showed that affinity chromatography could be an appropriate method for purification of IgG2b antibodies.