Murine Antibody Research Articles

Subtle Changes in the Combining Site of the Chlamydiaceae-Specific mAb S25-23 Increase the Antibody-Carbohydrate Binding Affinity by an Order of Magnitude.

Murine antibodies S25-23, S25-26, and S25-5 derive from a common germ-line origin, and all bind the Chlamydiaceae family-specific epitope αKdo(2→8)αKdo(2→4)αKdo (where Kdo is 3-deoxy-α-d- manno-oct-2-ulosonic acid) with high affinity and specificity. These antibodies recognize the entire trisaccharide antigen in a linkage-dependent manner via a groove composed largely of germ-line residues. Despite sharing identical heavy and light chain genes, S25-23 binds the family-specific epitope with nanomolar affinity, which is an order of magnitude higher than that of S25-26, while S25-5 displays an affinity between those of S25-23 and S25-26. We determined the high-resolution crystal structures of S25-23 and S25-5 antigen binding fragments in complex with a pentasaccharide derived from the LPS of Chlamydia and measured the affinity of S25-5 for chlamydial LPS antigens using isothermal titration microcalorimetry. The 1.75 Å resolution structure of S25-23 shows how subtle conservative mutations Arg(L)-27E to lysine and Ser(H)-56 to threonine lead to an order of magnitude increase in affinity. Importantly, comparison between previous S25-26 structures and the 1.99 and 2.05 Å resolution liganded and unliganded structures of S25-5, respectively, shows how a Ser(L)-27E mutation results in an intermediate affinity due to the reduced enthalpic penalty associated with complex formation that would otherwise be required for arginine in this position. This strategy allows for subtle adjustments in the combining site via affinity maturation that have dramatic consequences for the affinity of an antibody for its antigen.

Targeted Delivery of TNF Potentiates the Antibody-Dependent Cell-Mediated Cytotoxicity of an Anti-Melanoma Immunoglobulin

The recombinant murine IgG2a antibody TA99, directed against a melanoma antigen, was used to study combination modalities that potentiate antibody-dependent cell cytotoxicity. As previously reported, IgG2a(TA99) was extremely efficacious in preventing the growth of B16 lung metastases. However, the same antibody mediated only minimal tumor growth retardation when used to treat established neoplastic masses. The therapeutic activity of IgG2a(TA99) could be substantially enhanced by co-administration with an antibody-cytokine fusion (TA99-murine tumor necrosis factor [mTNF]), consisting of the TA99 antibody in single-chain variable fragment format fused to murine TNF. This fusion protein efficiently killed endothelial cells invitro and displayed only minimal activity against B16 melanoma cells. Invivo, TA99-mTNF boosted the influx of natural killer cells and macrophages into B16 melanoma lesions. Therapy studies with two different administration schedules showed that the combination of TA99-mTNF and IgG2a(TA99) was superior to the individual products used as single agents. The combination treatment converted most of the tumor mass into a necrotic lesion, but a vital tumor rim eventually regrew, even when dacarbazine was included in the therapeutic regimen. The treatment modality described in this article may be applicable to the treatment of melanoma patients, given the specificity of the gp75 antigen and its conservation across species.

Effects of the Humanized Anti-Adrenomedullin Antibody Adrecizumab (HAM8101) on Vascular Barrier Function and Survival in Rodent Models of Systemic Inflammation and Sepsis.

Adrenomedullin (ADM) is an important regulator of endothelial barrier function during sepsis. Administration of a murine antibody targeted against the N-terminus of ADM (HAM1101) resulted in improved outcome in models of murine sepsis. We studied the effects of a humanized form of this antibody (HAM8101, also known as Adrecizumab) on vascular barrier dysfunction and survival in rodent models of systemic inflammation and sepsis. Rats (n=48) received different dosages of HAM8101 or placebo (n = 8 per group), directly followed by administration of lipopolysaccharide (5 mg/kg). Twenty-four hours later, Evans Blue dye was administered to assess vascular leakage in kidney and liver tissue. Furthermore, mice (n = 24) were administered different dosages of HAM8101 or placebo (n = 6 per group), immediately followed by cecal ligation and puncture (CLP). Eighteen hours later, albumin, vascular endothelial growth factor (VEGF), and angiopoietin-1 were analyzed in the kidney. Finally, effects of single and repeated dose administration of HAM1101, HAM8101 and placebo on survival were assessed in CLP-induced murine sepsis (n = 60, n = 10 per group). Dosages of 0.1 and 2.5 mg/kg HAM8101 attenuated renal albumin leakage in endotoxemic rats. Dosages of 0.1, 2.0, and 20 mg/kg HAM8101 reduced renal concentrations of albumin and the detrimental protein VEGF in septic mice, whereas concentrations of the protective protein angiopoietin-1 were augmented. Both single and repeated administration of both HAM1101 and HAM8101 resulted in improved survival during murine sepsis. Pretreatment with the humanized anti-ADM antibody HAM8101 improved vascular barrier function and survival in rodent models of systemic inflammation and sepsis.

64cu-DOTA-Anti-CD33 PET-CT Imaging for Acute Myeloid Leukemia and Image-Guided Treatment

Introduction:Acute myeloid leukemia (AML) is a highly aggressive form of leukemia that results a poor survival outcome. Currently, diagnosis and prognosis are based on invasive single-point bone marrow biopsies (iliac crest). Although non-invasive positron emission tomography (PET) imaging has been developed for almost all solid tumors and some hematological malignancies, there is currently no non-invasive imaging specific to AML available, representing an unmet clinical need. About 85% of AML cells express CD33, and expression levels of CD33 has been correlated with poor survival outcomes (Pollard et al., Blood (2012)), making it an ideal candidate for immuno-PET. Therefore, our primary goal is to develop anti-CD33 immuno-PET for detecting CD33+ AML. The secondary goal is to assess the feasibility of CD33 PET image-guided external beam targeted radiation delivery in combination with chemotherapy (AraC).Methods:Murine anti-human CD33 monoclonal antibody clone p67.6 was conjugated to DOTA and radiolabeled with Cu-64 for imaging studies. In vivo PET-CT imaging and bio distribution of 64Cu-DOTA-anti-CD33 in vivo was carried out in NSG mice bearing CD33+ (MV4-11, HL60) AML cells. CD33-negative MM1s cells (multiple myeloma) were used as negative control. The AML and MM bearing mice were injected with 64Cu-DOTA-anti-CD33 (100 µCi/10 µg) and serial PET imaged at 24-48 h.We developed a functional total marrow irradiation (fTMI) treatment, where mice received total marrow irradiation (TMI) (2 Gy) and boost radiation (2 Gy) to regions with increased CD33 activity, followed by 2 days of AraC (40 mg/kg) and 24h later a bone marrow transplant. Furthermore, a humanized anti-human CD33 monoclonal antibody was generated and tested for immunogenicity against CD33 in AML cell lines and patient samples for future clinical studies.Results:PET-CT imaging and biodistribution studies of 64Cu-DOTA-anti-CD33 clearly indicates a CD33+ PET signal in the femur, tibia, humerus joints, L spine and spleen in AML-bearing mice, but not in multiple myeloma-bearing mice or in cold anti-CD33-DOTA treated leukemic mice (Figure 1A and B). Our new imaging method was able to detect CD33+ AML with a favorable sensitivity (71.4%) and specificity (100%). Based on detailed whole-body 3D imaging and validated with biodistribution studies, we discovered preferential regions in the skeletal system with differential CD33 activity, indicating the spatial heterogeneity of AML. CD33+ PET intensity was observed in the following descending order: femur≥lspine>humerus>tibia (Figure 1C).Next, using the PET-CT images, we targeted these CD33-active regions using the fTMI treatment plan. We have developed a unique irradiation system which allows targeting only skeletal tissues while sparing major organs like gut, lung and liver, unlike conventional total body irradiation (TBI). This approach will also provide an opportunity to escalate doses to specific regions in the body without damaging other unintended targets. The fTMI (2 Gy TMI and 2 Gy boost) treatment plan increased the medial survival of the mice to 43 days versus that of untreated control mice (26 days) or AraC treated mice (33 days) Figure(1D).Since the preclinical study was carried out using murine anti-CD33 antibody clone p67.6, we further tested the newly generated humanized anti-human-CD33 monoclonal antibody. This humanized antibody detects CD33 in both AML cell lines and human patient sample (Fig 1E).Conclusion:This study is the first to use an anti-CD33 monoclonal antibody for non-invasive immuno-PET-based imaging for AML detection, showing high sensitivity and specificity. This whole body imaging may be useful for AML diagnosis not only in the entire skeletal system, but also in the extramedullary organs, and for longitudinal monitoring of treatment response. Unlike conventional TBI, Image-guided fTMI along with conventional chemotherapy may result in an improved prognosis, as unintended major organs are spared from radiation. [Display omitted] DisclosuresVallera:GT Biopharma: Consultancy, Research Funding. Stein:Celgene: Speakers Bureau; Amgen Inc.: Speakers Bureau.

Targeting a Specific Glycosylated Epitope of CD43 with a New Humanized Monoclonal Antibody for the Treatment of Pediatric and Adult T-Cell Acute Lymphoblastic Leukemia (T-ALL)

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) accounts for about 20% of pediatric and adult ALL cases. Despite the use of intensive chemotherapy protocols, 25% of children and 50% of adult patients fail to respond or relapse. The 3-years prognosis for these patients is poor and novel treatment options are needed. The targeting of tumor-associated antigens by monoclonal antibodies (mAb) is among the most investigated immune-therapeutic strategies. Accordingly, we developed a new humanized mAb (hUMG1), directed against a heavy glycosylated epitope of CD43 which presents a high reactivity against T-ALL cells. Here we investigated the pre-clinical therapeutic activity and the mechanisms of action of hUMG1 in experimental models of T-ALL.Methods: The expression of hUMG1 target was assessed by flow cytometry on tumor cell lines and primary samples from either T-ALL patients (n=48) or healthy donors (n =6). Humanized mAbs were generated by combining the variable domains of the murine antibody to the corresponding human IgG1 constant domains. Through immunohistochemistry (IHC) we screened several tissue microarrays (TMA) including human, cynomolgusmonkey and macacus rhesus (according to FDA/CE guidelines), rat and mouse normal tissues. Complement-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and cellular phagocytosis (ADCP) on T-ALL cells were evaluated by flow cytometry. Six different in vivo models on NSG mice (3 with administration of NK-92-CD16+ effectors) have been generated to evaluate mAbs activity in different disease settings: orthotopic, subcutaneous advanced disease (treatments started at 100 mmc) and subcutaneous limited disease (treatments started the day after injection).Results: By screening different cancer cell lines, we observed hUMG1 target to be highly expressed on malignant T-ALL cells. We then tested T-ALL patient-derived blasts from 48 samples (40 pediatric and 8 adults) collected at diagnosis. The antigen was expressed on 23 out of 48 (48%), and most of them belonged to the subset of cortical (EGIL TIII) T-ALL group. By contrast the target antigen was not expressed on normal bone marrow cells from healthy donors. The analysis of the TMA including human normal tissues revealed a specific binding for thymus cortical lymphocytes only, leading us to hypothesize an acceptable safety profile. A humanized mAb, named hUMG1, and an afucosylated version of this mAb (aUMG1) were then developed. By gene expression profiling, western blot and flow cytometry, we observed that target binding by hUMG does not exert any direct activity on neoplastic cells. Subsequently, to investigate CDC, ADCC or ADCP, T-ALL cells were cultured in the presence of complement, peripheral blood mononuclear cells or macrophages, at increasing concentrations of both antibodies. Neither hUMG1 nor aUMG1 were able to induce CDC on target cells. Conversely, both mAbs induced CD16 downregulation, IFN-g production and degranulation on NK cells (more evident with aUMG1) and significant cytotoxicity against both T-ALL cell lines and primary blasts. Additionally, both mAbs induced ADCP. Lastly we observed a mAb-dependent activation of monocytes in the presence of target cells, as demonstrated by the reduction of CD16+ “non-classical” monocytes. Furthermore, we demonstrated potent activity of both mAbs in different T-ALL in vivo models. In an orthotopic model we observed 5 out of 20 treated mice free of disease after 100 days from injection as compared to none of the control group. In both subcutaneous models, we observed a strong ability of our antibody to delay tumor growth and to increase mice survival. Of note, the addition of NK-92-CD16+ strongly improved the activity of aUMG1. In the attempt to bring this antibody from bench to bedside, we assessed, through IHC, the expression of the hUMG1 target in healthy tissues from cynomolgus monkey, macacus rhesus, rat and mouse. We did not observe any reactivity, suggesting that the mAb target is very specific for human cells.Conclusion: Here, we demonstrated that hUMG1 mAb recognizes an antigen specifically expressed on the majority of T-ALL, and its binding is able to mediate effective cytotoxicity against leukemia in vitro and in vivo, indicating that this antibody, in particular the aUMG1 version, may represent a novel promising immune-therapeutic tool for the treatment of T-ALL patients. DisclosuresNo relevant conflicts of interest to declare.

The novel agonistic iNKT-cell antibody NKT14m induces a therapeutic antitumor response against B-cell lymphoma

ABSTRACTInvariant natural killer T (iNKT) cells are a small population of T lymphocytes that expresses an invariant T cell receptor with a unique specificity for glycolipid antigens. Their activation using the glycolipid α-galactosylceramide (α-GalCer) triggers innate and adaptive immune responses. The use of α-GalCer in preclinical models as a single antitumor treatment showed moderate effect, but its efficacy in cancer patients was less effective. In addition, this glycolipid induces long-term iNKT-cell anergy precluding the possibility of retreatment. Recently, the first murine iNKT-cell agonistic antibody, NKT14m, has been developed. Here, we analyzed, for the first time, the antitumor efficacy of NKT14m in a B-cell lymphoma model. In a therapeutic setting, a single dose of NKT14m had a moderate antitumor efficacy that was associated with an increase of IFN-γ producing iNKT cells even after a second dose of the NKT14m antibody. Importantly, the combination of a single dose of NKT14m with cyclophosphamide had a potent antitumor efficacy and long-lasting immunity in vivo. Our findings provide the first evidence of the in vivo antitumor efficacy of NKT14m antibody, showing that, either alone or in combination with chemotherapy, induces an effective antitumor response. These results open new opportunities for iNKT-cell mediated immunotherapy to treat B-cell lymphoma.

Biodistribution and Dosimetry of Indigenously Produced131I-Rituximab in B-Cell Lymphoma: Pilot Study Estimating Patient-Specific Dose Comparing 2 Different Dosimetric Methods

Cost containment through indigenous production of radioimmunotherapy agents for non-Hodgkin lymphoma (NHL) would be a pivotal step toward wider clinical availability, especially in developing countries. We examined the biodistribution and dosimetry of indigenously developed and radiolabeled 131I-rituximab, using the monoclonal antibody of chimeric origin, in patients with B-cell lymphoma for potential use in radioimmunotherapy. Methods: This prospective study included 13 patients with B-cell NHL who underwent low-dose diagnostic scanning for dosimetric and biodistribution studies. Soon after rituximab infusion, a diagnostic dose of radioiodinated rituximab was administered. Serial planar whole-body γ-camera images were taken soon afterward and on days 1, 2, 4, and 6. A source of 131I with known activity was used as a reference standard for dosimetry calculations. Results: The patient-specific administered dose that would give a whole-body absorbed radiation dose of 75 cGy, calculated by the MIRD schema, ranged from 3,095.42 to 6,330.33 MBq (83.66-171.09 mCi), with a mean of 3,986.01 ± 863.95 MBq (107.73 ± 23.35 mCi) and a median of 3,697.41 MBq (99.93 mCi). The mean residence time was 69.54 h. Within the first 48 h at least 50% of the injected activity was cleared, and by 144 h at least 80% was cleared. The patient-specific administered dose that would give a whole-body absorbed radiation dose of 75 cGy, calculated by mean residence time and activity-hours, ranged from 2,654.75 to 6,210.45 MBq (71.75-167.85 mCi), with a mean of 3,576.42 ± 927.59 MBq (96.66 ± 25.07 mCi) and a median of 3,421.02 MBq (92.46 mCi). With respect to organ-specific dosimetry, the mean absorbed doses to organs (apart from blood pool [3.77 Gy] and spleen [4.02 Gy]) were 0.97 Gy to the lungs, 0.69 Gy to the liver, and 0.7 Gy to the kidneys. Conclusion: The indigenous product had kinetics similar to commercial radiopharmaceuticals, with the advantage of a lower human antimouse antibody response because of the pharmaceutical's being a chimeric antibody rather than a murine antibody. Hence, clinical administration was safe. In none of the organs did dose-limiting radiation exposure occur at the proposed therapeutic dose.

Grasping at straws: the failure of solanezumab to modify mild Alzheimer’s disease

ABSTRACTIntroduction: The amyloid-beta (Aβ) cascade hypothesis is that reducing Aβ levels in the brain will be beneficial in the treatment of Alzheimer’s disease. Solanezumab is a humanized analog of a murine antibody that selectively targets the central domain of the soluble form of Aβ. In the EXPEDITION 1 and 2 Phase 3 clinical trials, solanezumab was shown to be ineffective in subjects with mild-to-moderate Alzheimer’s disease, and to have no effect on brain Aβ burden.Areas covered: This evaluation considers the secondary analysis of (EXPEDITION 1 and 2), which led to the EXPEDITION 3 trial of solanezumab in subjects with mild Alzheimer’s disease, and the results of EXPEDITION 3.Expert opinion: The secondary analysis of EXPEDITION 1 and 2 was limited to mild Alzheimer’s disease, and showed improvements on some scales, but not others. This analysis did not report data on Aβ burden. In my opinion, this was a questionable basis to undertake a further phase trial with solanezumab. The EXPEDITION 3 trial of solanezumab in subjects with mild Alzheimer’s disease was terminated early for ineffectiveness. With hindsight, solanezumab should have been discontinued after EXPEDITION 1 and 2, especially as it had not been shown to reduce Aβ burden.

PDGFRα monoclonal antibody: Assessment of embryo-fetal toxicity and time-dependent placental transfer of a murine surrogate antibody of olaratumab in mice.

Olaratumab (Lartruvo™) is a recombinant human IgG1 monoclonal antibody that specifically binds PDGFRα. The maternal and in utero embryo-fetal toxicity and toxicokinetics of a human anti-mouse PDGFRα antibody (LSN3338786) were investigated in pregnant mice. A pilot study was used to set doses for the definitive study. In the definitive study, mice were administered vehicle, 5, 50, or 150 mg/kg LSN3338786 by intravenous injection on gestation days (GD) 6, 9, 12, and 15. Fetal tissues and/or serum samples were collected on GD 10, 12, 15, and 18 to evaluate exposure of antibody. There were no adverse maternal effects at 50 and 150 mg/kg although maternal deaths and adverse clinical signs were observed at 5 mg/kg. LSN3338786 crossed the placenta as early as GD 10 during organogenesis. Elimination half-life of LSN3338786 in dams decreased between GD 6 and 15. On GD 18, fetal serum concentrations of antibody were substantially higher than maternal serum concentrations at all doses. Increased incidences of malformations consisting of open and partially open eye and increased incidences of skeletal variation frontal/parietal additional ossification site occurred in fetuses from mid- and high-dose groups. The majority of transplacental migration of antibody occurred in concert with rapid maternal serum clearance before parturition. The no-observed effect level for teratogenicity of 5 mg/kg was associated with GD 15 maternal serum concentrations 3-11 times lower than clinical exposure of olaratumab, suggesting that olaratumab may cause fetal harm when administered to pregnant women.

A comparison of unamplified and massively multiplexed PCR amplification for murine antibody repertoire sequencing.

Sequencing antibody repertoires has steadily become cheaper and easier. Sequencing methods usually rely on some form of amplification, often a massively multiplexed PCR prior to sequencing. To eliminate potential biases and create a data set that could be used for other studies, our laboratory compared unamplified sequencing results from the splenic heavy‐chain repertoire in the mouse to those processed through two commercial applications. We also compared the use of mRNA vs total RNA, reverse transcriptase, and primer usage for cDNA synthesis and submission. The use of mRNA for cDNA synthesis resulted in higher read counts but reverse transcriptase and primer usage had no statistical effects on read count. Although most of the amplified data sets contained more antibody reads than the unamplified data set, we detected more unique variable (V)‐gene segments in the unamplified data set. Although unique CDR3 detection was much lower in the unamplified data set, RNASeq detected 98% of the high‐frequency CDR3s. We have shown that unamplified profiling of the antibody repertoire is possible, detects more V‐gene segments, and detects high‐frequency clones in the repertoire.

Structure of the Recombinant Neisseria gonorrhoeae Adhesin Complex Protein (rNg-ACP) and Generation of Murine Antibodies with Bactericidal Activity against Gonococci.

Neisseria gonorrhoeae (gonococcus [Ng]) is the causative organism of the sexually transmitted disease gonorrhoea, and no effective vaccine exists currently. In this study, the structure, biological properties, and vaccine potential of the Ng-adhesin complex protein (Ng-ACP) are presented. The crystal structure of recombinant Ng-ACP (rNg-ACP) protein was solved at 1.65 Å. Diversity and conservation of Ng-ACP were examined in different Neisseria species and gonococcal isolates (https://pubmlst.org/neisseria/ database) in silico, and protein expression among 50 gonococcal strains in the Centers for Disease Control and Prevention/Food and Drug Administration (CDCP/FDA) AR Isolate Bank was examined by Western blotting. Murine antisera were raised to allele 10 (strain P9-17)-encoded rNg-ACP protein with different adjuvants and examined by enzyme-linked immunosorbent assay (ELISA), Western blotting, and a human serum bactericidal assay. Rabbit antiserum to rNg-ACP was tested for its ability to prevent Ng-ACP from inhibiting human lysozyme activity in vitro. Ng-ACP is structurally homologous to Neisseria meningitidis ACP and MliC/PliC lysozyme inhibitors. Gonococci expressed predominantly allele 10- and allele 6-encoded Ng-ACP (81% and 15% of isolates, respectively). Murine antisera were bactericidal (titers of 64 to 512, P < 0.05) for the homologous P9-17 strain and heterologous (allele 6) FA1090 strain. Rabbit anti-rNg-ACP serum prevented Ng-ACP from inhibiting human lysozyme with ∼100% efficiency. Ng-ACP protein was expressed by all 50 gonococcal isolates examined with minor differences in the relative levels of expression. rNg-ACP is a potential vaccine candidate that induces antibodies that (i) are bactericidal and (ii) prevent the gonococcus from inhibiting the lytic activity of an innate defense molecule.IMPORTANCENeisseria gonorrhoeae (gonococcus [Ng]) is the causative organism of the sexually transmitted disease gonorrhoea, and the organism is listed by the World Health Organization as a high-priority pathogen for research and development of new control measures, including vaccines. In this study, we demonstrated that the N. gonorrhoeae adhesin complex protein (Ng-ACP) was conserved and expressed by 50 gonococcal strains and that recombinant proteins induced antibodies in mice that killed the bacteria in vitro We determined the structure of Ng-ACP by X-ray crystallography and investigated structural conservation with Neisseria meningitidis ACP and MliC/PliC proteins from other bacteria which act as inhibitors of the human innate defense molecule lysozyme. These findings are important and suggest that Ng-ACP could provide a potential dual target for tackling gonococcal infections.

Computational B-cell epitope identification and production of neutralizing murine antibodies against Atroxlysin-I

Epitope identification is essential for developing effective antibodies that can detect and neutralize bioactive proteins. Computational prediction is a valuable and time-saving alternative for experimental identification. Current computational methods for epitope prediction are underused and undervalued due to their high false positive rate. In this work, we targeted common properties of linear B-cell epitopes identified in an individual protein class (metalloendopeptidases) and introduced an alternative method to reduce the false positive rate and increase accuracy, proposing to restrict predictive models to a single specific protein class. For this purpose, curated epitope sequences from metalloendopeptidases were transformed into frame-shifted Kmers (3 to 15 amino acid residues long). These Kmers were decomposed into a matrix of biochemical attributes and used to train a decision tree classifier. The resulting prediction model showed a lower false positive rate and greater area under the curve when compared to state-of-the-art methods. Our predictions were used for synthesizing peptides mimicking the predicted epitopes for immunization of mice. A predicted linear epitope that was previously undetected by an experimental immunoassay was able to induce neutralizing-antibody production in mice. Therefore, we present an improved prediction alternative and show that computationally identified epitopes can go undetected during experimental mapping.

Abstract A30: Novel T-cell engaging antibodies against L1CAM for neuroblastoma

Abstract Background: T cell-mediated immunotherapy has shown great promise in the treatment of human cancers. Bispecific antibodies (BsAbs) that engage CD3 on T cells and a tumor antigen on target cells can induce polyclonal T cell-mediated cytotoxicity against both leukemia (expressing CD19) and solid tumors (expressing HER-2 or GD2), unrestricted by MHC. The L1 cell adhesion molecule (L1CAM) is a glycoprotein consisting of 6 Ig-like domains and 5 fibronectin-like repeats in the ectodomain. In normal tissues, L1CAM expression is restricted to neurons, renal tubules, and skin at low levels and plays an important role in brain development, including neuronal migration and differentiation. Interestingly, L1CAM is also overexpressed on various pediatric cancers including neuroblastoma (NB), and stimulates tumor proliferation, migration, and metastasis. Chimeric IgG1 antibody, chCE7, targeting the 6th Ig-like domain of human L1CAM, had high affinity (KD = 0.63 nM) and when 131I-labeled, had superior sensitivity and specificity over 131I-MIBG in patients with metastatic NB. However, chCE7 lacked ADCC ability in preclinical studies and no major clinical responses have been reported for anti-L1CAM chimeric antigen receptor (CAR) modified T cells (NCT02311621). Methods: By CDR grafting, the murine antibody E71 (specific for 2nd Ig-like domain with KD = 2.23 nM) was humanized to huE71, and huE71-BsAb using the IgG(L)-scFv BsAb platform previously described (Xu et al., Can Immunol Res 2016) was generated and compared to huE72 (the humanized version of CE7)-BsAb. The BsAbs were produced in CHO-S cells and purified by protein A affinity chromatography. Size homogeneity was confirmed by SEC HPLC. Binding to L1CAM and CD3, or to tumors and T cells was measured by SPR (Biacore) or by FACS, respectively. In vitro T-cell activation and cytokine release were assessed by FACS and by ELISA, respectively. In vitro tumor cytotoxicity was measured by 4-hour 51Cr release at an E:T ratio of 10:1. In vivo antitumor effect was assessed in Balb/c-Rag2-/-IL-2R-c-KO (DKO) mice xenografted with subcutaneous (s.c.) NB cell lines or patient-derived xenografts (PDX). Tumor targeting was performed by using 89Zr-labeled huE71 and huE72 IgG1 in xenografted nude mice. Results: Both BsAbs showed a molecular size of 210kDa with &amp;gt;90% purity. When assessed by SPR for binding to the L1CAM, huE71-BsAb retained its parental affinity, while that of huE72-BsAb decreased by 8-fold (KD of 5.15nM and 5.28nM, respectively). As expected from the IgG(L)-scFv design, both BsAbs showed lower binding to CD3(+) T cells when compared with huOKT3 IgG1. Both BsAbs induced CD25 expression on CD4(+) or CD8(+) T cells after 96 hours of culture in the presence of L1CAM(+) IMR32 NB cell line, and CD8(+) T cells showed more activation in the presence of huE72-BsAb. Both BsAbs induced similar level of Th1 cytokine (e.g. TNFα) from PBMCs following 24 hours of culture in the presence of IMR32. Both BsAbs mediated T-cell cytotoxicity on L1CAM(+) cell lines with pM EC50s. In nude mice xenografted with s.c. L1CAM(+) cancer cell line, intravenous (i.v.) huE71 IgG1 showed higher tumor targeting efficiency than huE72 IgG1. In DKO mice xenografted with s.c. IMR32 (carrying luciferase reporter gene) and s.c. PBMCs, i.v. huE71-BsAb significantly suppressed tumor growth while huE72-BsAb had no antitumor effect. Similar results were observed in an s.c. L1CAM(+) NB PDX model when PBMCs were injected i.v. Conclusions: Despite near-identical in vitro binding characteristics, BsAbs targeting different epitopes on the same target have strikingly different antitumor properties in vivo. HuE71-BsAb is a potential T cell engaging immunotherapeutic for NB and other L1CAM(+) solid tumors. Citation Format: Maya Suzuki, Hong Xu, Hongfen Guo, Brandon Nemieboka, Zhihao Wu, Jason Lewis, Nai-Kong V. Cheung. Novel T-cell engaging antibodies against L1CAM for neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A30.

Development of a Tetravalent Anti-GPA33/Anti-CD3 Bispecific Antibody for Colorectal Cancers.

Despite progress in the treatment of colorectal cancer, curing metastatic colorectal cancer remains a major unmet medical need worldwide. Here, we describe a T-cell-engaging bispecific antibody (T-BsAb) to redirect polyclonal cytotoxic T cells to eradicate colorectal cancer. A33, a murine antibody specific for GPA33, was humanized to huA33 and reformatted to huA33-BsAb, based on a novel IgG(L)-scFv platform by linking the anti-CD3 huOKT3 scFv to the carboxyl end of the light chain. This T-BsAb was stably expressed in CHO cells and purified as a stable monomer by HPLC, retaining immunoreactivity by FACS through 30 days of incubation at 37°C. In vitro, it induced activation and expansion of unstimulated T cells and elicited potent T-cell-dependent cell-mediated cytotoxicity against colon and gastric cancer cells in an antigen-specific manner. In vivo, huA33-BsAb inhibited the colon and gastric cancer xenografts, in both subcutaneous and intraperitoneal tumor models. More importantly, both microsatellite instable and microsatellite stable colorectal cancer were effectively eliminated by huA33-BsAb. These preclinical results provide further support for the use of IgG(L)-scFv platform to build BsAb, and especially one targeting GPA33 for colorectal cancer. These preclinical results also support further development of huA33-BsAb as a potential immunotherapeutic. Mol Cancer Ther; 17(10); 2164-75. ©2018 AACR.

Humanized 3F8 Anti-GD2 Monoclonal Antibody Dosing With Granulocyte-Macrophage Colony-Stimulating Factor in Patients With Resistant Neuroblastoma

Chimeric and murine anti-GD2 antibodies are active against neuroblastoma, but the development of neutralizing antibodies can compromise efficacy. To decrease immunogenicity, hu3F8, a humanized anti-GD2 antibody, was constructed. To find the maximum-tolerated dose of hu3F8 with granulocyte-macrophage colony-stimulating factor. This phase 1 clinical trial used a 3 + 3 dose-escalation design in a single referral center (Memorial Sloan Kettering Cancer Center, New York, New York). Participants were enrolled from December 24, 2012, through May 3, 2016, with follow-up and analyses through February 28, 2018. Eligibility criteria included older than 1 year and resistant or recurrent neuroblastoma regardless of the number or kinds of prior treatments. All 57 participants met the eligibility criteria, received treatment according to the protocol, and were included in all analyses. Treatment cycles were monthly, if human antihuman antibody remained negative. Each cycle comprised hu3F8 infused intravenously for 30 minutes on Monday, Wednesday, and Friday as well as granulocyte-macrophage colony-stimulating factor administered subcutaneously daily from 5 days before infusion through the last day of infusion. After cycle 2, hu3F8 was increased to the highest dose level that had been confirmed as safe. Toxicity, pharmacokinetics, immunogenicity, and disease response. Of the 57 participants, 34 (60%) were male and 23 (40%) were female (male-to-female ratio of 1.5), with a median (range) age of 6.8 (2.4-31.3) years at enrollment and a median (range) time of 3.1 (0.6-9.0) years since initial chemotherapy. Participants received a median (range) of 4 (1-15) cycles. Treatment was outpatient with reversible neuropathic pain and without unexpected toxic effects. No maximum-tolerated dose was identified. Dose escalation was associated with increased serum levels and proceeded through dosage of 9.6 mg/kg/cycle (approximately 288 mg/m2), which is more than 2.5 times higher than the standard dosage of 75 mg/m2/cycle or 100 mg/m2/cycle of dinutuximab and m3F8. Human antihuman antibody positivity developed in 5 of 57 patients (9%) after cycle 1, including in 1 of 10 patients (10%) not previously treated with anti-GD2 antibody and in 4 of 47 patients (9%) previously exposed to 1 or 2 anti-GD2 antibodies. Antineuroblastoma activity included major responses associated with higher dosing and prolonged progression-free survival despite a history of relapses. This phase 1 clinical trial found hu3F8 to be associated with modest toxic effects, low immunogenicity, and substantial antineuroblastoma activity; phase 2 trials are in progress. ClinicalTrials.gov identifier: NCT01757626.

Tropomyosins in mosquito and house dust mite cross‐react at the humoral and cellular level

Aedes aegypti and Dermatophagoides pteronyssinus contain important allergens including cross-reactive tropomyosins. However, the functional and clinical relevance of their cross-reactivity is still debated. To analyse the humoral and cellular cross-reactivity of recombinant Aed a 10.01, Aed a 10.02 and Der p 10. Sera from 15 Austrian house dust mite-allergic, Der p 10-sensitized individuals were tested for IgE reactivity to recombinant tropomyosins in ELISA, inhibition ELISA and basophil activation tests. BALB/c mice were immunized with Aed a 10.01 or Aed a 10.02, and their sera were assessed for reactivity to all tropomyosins. Splenocytes were stimulated with all tropomyosins and synthetic peptides representing the amino acid sequence of Aed a 10.01. IgE antibodies of Der p 10-sensitized patients cross-reacted with both tropomyosins from A.aegypti. Aed a 10.01 was a more potent inhibitor of IgE binding to Der p 10 and a stronger activator of basophils sensitized with Der p 10-specific IgE than Aed a 10.02. Murine antibodies raised against Aed a 10.01 and Aed a 10.02 cross-reacted with Der p 10. Aed a 10.01-specific antibody showed stronger cross-reactivity with Der p 10 than Aed a 10.02-specific antibody. Splenocytes from both groups of mice proliferated similarly to all tropomyosins. Five cross-reactive T cell-activating regions were identified. Tropomyosins from D.pteronyssinus and A.aegypti show humoral and cellular cross-reactivity, involving 5 potential T cell-activating regions. The more pronounced cross-reactivity of Aed a 10.01 and Der p 10 matched the higher sequence similarity of both proteins.

Anti-CD52 antibody treatment depletes B cell aggregates in the central nervous system in a mouse model of multiple sclerosis

BackgroundMultiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) for which several new treatment options were recently introduced. Among them is the monoclonal anti-CD52 antibody alemtuzumab that depletes mainly B cells and T cells in the immune periphery. Considering the ongoing controversy about the involvement of B cells and in particular the formation of B cell aggregates in the brains of progressive MS patients, an in-depth understanding of the effects of anti-CD52 antibody treatment on the B cell compartment in the CNS itself is desirable.MethodsWe used myelin basic protein (MBP)-proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 (B6) mice as B cell-dependent model of MS. Mice were treated intraperitoneally either at the peak of EAE or at 60 days after onset with 200 μg murine anti-CD52 vs. IgG2a isotype control antibody for five consecutive days. Disease was subsequently monitored for 10 days. The antigen-specific B cell/antibody response was measured by ELISPOT and ELISA. Effects on CNS infiltration and B cell aggregation were determined by immunohistochemistry. Neurodegeneration was evaluated by Luxol Fast Blue, SMI-32, and Olig2/APC staining as well as by electron microscopy and phosphorylated heavy neurofilament serum ELISA.ResultsTreatment with anti-CD52 antibody attenuated EAE only when administered at the peak of disease. While there was no effect on the production of MP4-specific IgG, the treatment almost completely depleted CNS infiltrates and B cell aggregates even when given as late as 60 days after onset. On the ultrastructural level, we observed significantly less axonal damage in the spinal cord and cerebellum in chronic EAE after anti-CD52 treatment.ConclusionAnti-CD52 treatment abrogated B cell infiltration and disrupted existing B cell aggregates in the CNS.

Construction, Expression, and Characterization of a Novel Human-Mouse Chimeric Antibody, Hm3A4: A Potential Therapeutic Agent for B and Myeloid Lineage Leukemias.

Antibody-targeting therapy has drawn great interests to the hematologists and oncologists. 3A4, a novel antibody recognizing human CD45RA antigen, is a new target molecule for leukemias and holds a therapeutic potential for myeloid lineage leukemias. However, murine antibodies cannot be safely used in patients because of their strong immune reaction, humanization of the antibodies interested will be an important development step for therapeutic purpose. The aim of this study was to engineer the mouse 3A4 and to investigate the biological activity of its chimeric form. The humanized antibody composed of the 3A4 single-chain fragment of variable region and the human IgG1 Fc region, which was named human-mouse chimeric antibody 3A4 (Hm3A4). The function and biological activities of Hm3A4 were characterized using a variety of biological approaches. The results showed that Hm3A4 retained a strong binding activity to its antigen and could significantly block the binding of parental 3A4 to the antigen. In vitro experiments revealed that Hm3A4 could kill the target cells through complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity function. In vivo, Hm3A4 showed efficient antileukemia activity outperforming the nontreated mice. In conclusion, the chimeric antibody has an excellent biological activity after humanization and holds targeting therapeutic potential for myeloid leukemia, which warrants further development of this agent.

ASAP - A Webserver for Immunoglobulin-Sequencing Analysis Pipeline.

Reproducible and robust data on antibody repertoires are invaluable for basic and applied immunology. Next-generation sequencing (NGS) of antibody variable regions has emerged as a powerful tool in systems immunology, providing quantitative molecular information on antibody polyclonal composition. However, major computational challenges exist when analyzing antibody sequences, from error handling to hypermutation profiles and clonal expansion analyses. In this work, we developed the ASAP (A webserver for Immunoglobulin-Seq Analysis Pipeline) webserver (https://asap.tau.ac.il). The input to ASAP is a paired-end sequence dataset from one or more replicates, with or without unique molecular identifiers. These datasets can be derived from NGS of human or murine antibody variable regions. ASAP first filters and annotates the sequence reads using public or user-provided germline sequence information. The ASAP webserver next performs various calculations, including somatic hypermutation level, CDR3 lengths, V(D)J family assignments, and V(D)J combination distribution. These analyses are repeated for each replicate. ASAP provides additional information by analyzing the commonalities and differences between the repeats (“joint” analysis). For example, ASAP examines the shared variable regions and their frequency in each replicate to determine which sequences are less likely to be a result of a sample preparation derived and/or sequencing errors. Moreover, ASAP clusters the data to clones and reports the identity and prevalence of top ranking clones (clonal expansion analysis). ASAP further provides the distribution of synonymous and non-synonymous mutations within the V genes somatic hypermutations. Finally, ASAP provides means to process the data for proteomic analysis of serum/secreted antibodies by generating a variable region database for liquid chromatography high resolution tandem mass spectrometry (LC-MS/MS) interpretation. ASAP is user-friendly, free, and open to all users, with no login requirement. ASAP is applicable for researchers interested in basic questions related to B cell development and differentiation, as well as applied researchers who are interested in vaccine development and monoclonal antibody engineering. By virtue of its user-friendliness, ASAP opens the antibody analysis field to non-expert users who seek to boost their research with immune repertoire analysis.

An in vitro paradigm to assess potential anti-A\u03b2 antibodies for Alzheimer\u2019s disease

Although the amyloid β-protein (Aβ) is believed to play an initiating role in Alzheimer’s disease (AD), the molecular characteristics of the key pathogenic Aβ forms are not well understood. As a result, it has proved difficult to identify optimal agents that target disease-relevant forms of Aβ. Here, we combined the use of Aβ-rich aqueous extracts of brain samples from AD patients as a source of human Aβ and live-cell imaging of iPSC-derived human neurons to develop a bioassay capable of quantifying the relative protective effects of multiple anti-Aβ antibodies. We report the characterization of 1C22, an aggregate-preferring murine anti-Aβ antibody, which better protects against forms of Aβ oligomers that are toxic to neurites than do the murine precursors of the clinical immunotherapeutics, bapineuzumab and solanezumab. These results suggest further examination of 1C22 is warranted, and that this bioassay maybe useful as a primary screen to identify yet more potent anti-Aβ therapeutics.