781 GT-001 - anti-Lewis Y antibody with superior fine-specificity and reduced off-target binding

BackgroundThe Lewis Y (CD174) carbohydrate antigen is widely expressed in primary and metastatic epithelial tumors like colon, lung, ovarian, and breast. Targeting Lewis Y for cancer therapy was pursued before, however, other anti-Lewis Y antibodies tested in clinical trials showed cross-reactivity to related carbohydrate structures expressed on blood cells and mostly failed for efficacy and/or safety reasons.1–4 We have developed a humanized antibody (GT-001) that shows superior fine-specificity and higher affinity compared to clinically tested anti-Lewis Y antibodies BR96 and h3S193.MethodsThe specificity and cross-reactivity of GT-001, BR96 and h3S193 were compared. Cross-reactivity binding to related carbohydrate PAA-conjugates was tested via ELISA and affinity towards Lewis Y-PAA was measured using switchSENSE® technology (DRX2, Dynamic Biosensors). Functional binding to several tumor cell lines and healthy human leukocytes was analyzed via flow cytometry. Binding of GT-001 to different cancer indications was analyzed by immunohistochemistry. Inhibition of tumor cell proliferation was tested using GT-001 coupled to ProtG-MMAE.ResultsGT-001 is strictly specific for Lewis Y and does not cross-react with >90 related carbohydrate structures tested. Our lead candidate shows superior fine-specificity compared to BR96, for which we could confirm the reported cross-reactivity towards Lewis X,5 and stronger binding of Lewis Y compared to h3S193 as shown by affinity measurement. Further, GT-001 shows no/weak binding to blood cells whereas BR96 and h3S193 significantly bind to different leukocyte subsets. IHC studies reveal that GT-001 stains tumor tissue of different cancer indications (breast cancer, colorectal cancer, head and neck cancer, (non) small cell lung cancer and ovarian cancer) at a high percentage of cases. In ADC surrogate assays, GT-001 potently inhibits the proliferation of several tumor cell lines indicating effective internalization.ConclusionsLewis Y is expressed on many epithelial tumor indications of high medical need. However, several approaches of targeting Lewis Y have failed in the past for efficacy and/or safety reasons. We have developed a humanized antibody that shows superior fine-specificity and higher affinity compared to clinically tested anti-Lewis Y antibodies BR96 and h3S193. Due to the superior fine-specificity, GT-001 shows no/reduced binding of healthy leukocytes potentially reducing side effects as observed for BR96 in the clinic. Its strong target binding and internalization properties make GT-001 an ideal candidate for ADC development.ReferencesAjani JA, Kelsen DP, Haller D, Hargraves K, Healey D. A multi-institutional phase II study of BMS-182248-01 (BR96-doxorubicin conjugate) administered every 21 days in patients with advanced gastric adenocarcinoma. Cancer J 2000;6(2):78–81.Saleh MN, Sugarman S, Murray J, Ostroff JB, Healey D, Jones D, Daniel CR, LeBherz D, Brewer H, Onetto N, LoBuglio AF. Phase I trial of the anti-Lewis Y drug immunoconjugate BR96-doxorubicin in patients with lewis Y-expressing epithelial tumors. J Clin Oncol 2000;18(11):2282–92.Scott AM, Tebbutt N, Lee FT, Cavicchiolo T, Liu Z, Gill S, Poon AM, Hopkins W, Smyth FE, Murone C, MacGregor D, Papenfuss AT, Chappell B, Saunder TH, Brechbiel MW, Davis ID, Murphy R, Chong G, Hoffman EW, Old LJ. A phase I biodistribution and pharmacokinetic trial of humanized monoclonal antibody Hu3s193 in patients with advanced epithelial cancers that express the Lewis-Y antigen. Clin Cancer Res 2007;13(11):3286–92.Smaletz O, Diz MD, do Carmo CC, Sabbaga J, Cunha-Junior GF, Azevedo SJ, Maluf FC, Barrios CH, Costa RL, Fontana AG, Madrigal V, Wainstein AJ, Yeda FP, Alves VA, Moro AM, Blasbalg R, Scott AM, Hoffman EW. A phase II trial with anti-Lewis-Y monoclonal antibody (hu3S193) for the treatment of platinum resistant/refractory ovarian, fallopian tube and primary peritoneal carcinoma. Gynecol Oncol 2015;138(2):272–7.Zhang S, Zhang HS, Cordon-Cardo C, Reuter VE, Singhal AK, Lloyd KO, Livingston PO. Selection of tumor antigens as targets for immune attack using immunohistochemistry: II. Blood group-related antigens. Int J Cancer 1997;73(1):50–6.

Hinge Influences in Murine IgG Binding to Cryptococcus neoformans Capsule

Decades of studies on antibody structure led to the tenet that the V region binds antigens while the C region interacts with immune effectors. In some antibodies, however, the C region affects affinity and/or specificity for the antigen. One such case is that of the 3E5 antibodies, a family of monoclonal murine IgGs in which the mIgG3 isotype has different fine specificity to the Cryptococcus neoformans capsule polysaccharide than the other mIgG isotypes. Our group serendipitously found another pair of mIgG1/mIgG3 antibodies based on the 2H1 hybridoma to the C. neoformans capsule that recapitulated the differences observed with 3E5. In this work, we report the molecular basis of the constant domain effects on antigen binding using recombinant antibodies. As with 3E5, immunofluorescence experiments show a punctate pattern for 2H1-mIgG3 and an annular pattern for 2H1-mIgG1. Also as observed with 3E5, 2H1-mIgG3 bound on ELISA to both acetylated and non-acetylated capsular polysaccharide, whereas 2H1-mIgG1 only bound well to the acetylated form, consistent with differences in fine specificity. In engineering hybrid mIgG1/mIgG3 antibodies, we found that switching the 2H1-mIgG3 hinge for its mIgG1 counterpart changed the immunofluorescence pattern to annular, but a 2H1-mIgG1 antibody with a mIgG3 hinge still had an annular pattern. The hinge is thus necessary but not sufficient for these changes in binding to the antigen. This important role for the constant region in binding of antibodies to the antigen could affect the design of therapeutic antibodies and our understanding of their function in immunity.Key pointsKey point 1- 2H1 antibodies recapitulate differences between mIgG isotypes observed with 3E5.Key point 2 – The hinge region is necessary but not sufficient for these differences.Key point 3 - The antibody constant region can also play a role in mIgG binding to antigen.

Identification of a neutralizing epitope within minor repeat region of Plasmodium falciparum CS protein

Malaria remains a major cause of morbidity and mortality worldwide with 219 million infections and 435,000 deaths predominantly in Africa. The infective Plasmodium sporozoite is the target of a potent humoral immune response that can protect murine, simian and human hosts against challenge by malaria-infected mosquitoes. Early murine studies demonstrated that sporozoites or subunit vaccines based on the sporozoite major surface antigen, the circumsporozoite (CS) protein, elicit antibodies that primarily target the central repeat region of the CS protein. In the current murine studies, using monoclonal antibodies and polyclonal sera obtained following immunization with P. falciparum sporozoites or synthetic repeat peptides, we demonstrate differences in the ability of these antibodies to recognize the major and minor repeats contained in the central repeat region. The biological relevance of these differences in fine specificity was explored using a transgenic P. berghei rodent parasite expressing the P. falciparum CS repeat region. In these in vitro and in vivo studies, we demonstrate that the minor repeat region, comprised of three copies of alternating NANP and NVDP tetramer repeats, contains an epitope recognized by sporozoite-neutralizing antibodies. In contrast, murine monoclonal antibodies specific for the major CS repeats (NANP)n could be isolated from peptide-immunized mice that had limited or no sporozoite-neutralizing activity. These studies highlight the importance of assessing the fine specificity and functions of antirepeat antibodies elicited by P. falciparum CS-based vaccines and suggest that the design of immunogens to increase antibody responses to minor CS repeats may enhance vaccine efficacy.

Hierarchy in the Avidity and Cross-reactivity of Anti-citrullinated Protein Antibodies in the Serum of Patients With Rheumatoid Arthritis

BackgroundFine specificity of anti-citrullinated protein antibodies (ACPAs), in which cross-reactivity exists, varies among patients with rheumatoid arthritis (RA), but it is unclear whether the mechanism of ACPA production is same or different among individuals. Since avidity of serum antibody reflects the direction of immune response, we compared the levels of avidity and cross-reactivity between various ACPAs in a cohort of RA patient.MethodsSera from 180 RA patients positive for anti-cyclic citrullinated peptide (CCP) 2 antibody were screened for positivity of antibodies against CCP1, and citrullnated fibrinogen (cFib), enolase (cEno), and vimentin (cVim) peptides. Avidity of the four ACPAs, and some autoantibodies and antibodies against foreign antigens was determined by an elution assay using sodium thiocyanate solution. Cross-reactivity between different ACPAs was estimated by measuring the inhibition of binding by competitor peptides. ResultsThe prevalence of anti-CCP1, anti-cFib, anti-cEno, and anti-cVim antibodies in the anti-CCP2-positive RA cohort were 37.7%, 38.3%, 15.6%, and 23.9%, respectively. The avidity of ACPAs, except for anti-cVim antibody, was significantly lower than that of antibodies against foreign antigens, while there was a large variety in the avidity of other autoantibodies. At individual levels, the avidity of anti-cVim was significantly higher than that of other ACPAs, and there was a significant correlation in the avidity of anti-CCP and anti-cFib antibodies. Substantial extent of cross-reactivity was seen between different ACPAs, which also showed a fixed hierarchy.ConclusionThe fixed hierarchy in the avidity and cross-reactivity between different ACPAs suggests that the mechanism underlying ACPA production is common to all RA patients. Presence of a dominant antigen that induces whole ACPA response is speculated.

Rare Detection of Antiviral Functions of Polyclonal IgA Isolated from Plasma and Breast Milk Compartments in Women Chronically Infected with HIV-1

ABSTRACTThe humoral response to invading mucosal pathogens comprises multiple antibody isotypes derived from systemic and mucosal compartments. To understand the contribution of each antibody isotype/source to the mucosal humoral response, parallel investigation of the specificities and functions of antibodies within and across isotypes and compartments is required. The role of IgA against HIV-1 is complex, with studies supporting a protective role as well as a role for serum IgA in blocking effector functions. Thus, we explored the fine specificity and function of IgA in both plasma and mucosal secretions important to infant HIV-1 infection, i.e., breast milk. IgA and IgG were isolated from milk and plasma from 20 HIV-1-infected lactating Malawian women. HIV-1 binding specificities, neutralization potency, inhibition of virus-epithelial cell binding, and antibody-mediated phagocytosis were measured. Fine-specificity mapping showed IgA and IgG responses to multiple HIV-1 Env epitopes, including conformational V1/V2 and linear V2, V3, and constant region 5 (C5). Env IgA was heterogeneous between the milk and systemic compartments (Env IgA, τ = 0.00 to 0.63,P= 0.0046 to 1.00). Furthermore, IgA and IgG appeared compartmentalized as there was a lack of correlation between the specificities of Env-specific IgA and IgG (in milk, τ = −0.07 to 0.26,P= 0.35 to 0.83). IgA and IgG also differed in functions: while neutralization and phagocytosis were consistently mediated by milk and plasma IgG, they were rarely detected in IgA from both milk and plasma. Understanding the ontogeny of the divergent IgG and IgA antigen specificity repertoires and their effects on antibody function will inform vaccination approaches targeted toward mucosal pathogens.IMPORTANCEAntibodies within the mucosa are part of the first line of defense against mucosal pathogens. Evaluating mucosal antibody isotypes, specificities, and antiviral functions in relationship to the systemic antibody profile can provide insights into whether the antibody response is coordinated in response to mucosal pathogens. In a natural immunity cohort of HIV-infected lactating women, we mapped the fine specificity and function of IgA in breast milk and plasma and compared these with the autologous IgG responses. Antigen specificities and functions differed between IgG and IgA, with antiviral functions (neutralization and phagocytosis) predominantly mediated by the IgG fraction in both milk and plasma. Furthermore, the specificity of milk IgA differed from that of systemic IgA. Our data suggest that milk IgA and systemic IgA should be separately examined as potential correlates of risk. Preventive vaccines may need to employ different strategies to elicit functional antiviral immunity by both antibody isotypes in the mucosa.