Human IgG1 Isotype Research Articles

CD20 Antibodies of Human IgA Isotype Mediate CDC, and ADCC By Myeloid Effector Cells

Introduction: Three unconjugated CD20 antibodies have been approved for the treatment of lymphoma patients. All three are of human IgG1 isotype, but nevertheless they differ in their modes of action: type I antibodies (e.g. rituximab and ofatumumab) trigger effective complement-dependent cytotoxicity (CDC), whereas type II antibodies (e.g. obinutuzumab) are potent inducers of direct cell death, while both type I and II antibodies can induce antibody-dependent cellular cytotoxicity (ADCC). Studies in syngeneic mouse models suggest that myeloid cells are the predominant effector cell type for CD20 antibodies (Uchida et al. J Exp Med 199:1659, 2004). However, human myeloid cells, particularly PMN, are activated more effectively by human IgA than by IgG1 antibodies (Dechant et al. Blood 100:4574, 2002) - especially when the latter are engineered for enhanced FcγRIII affinity (Peipp et al. Blood 112:2390, 2008). Antibodies of IgA isotype constitute an integral part of the mucosal immune system, and differ from IgG antibodies in their pharmacokinetic properties and immune effector mechanisms (Boross et al. EMBO Mol Med 5:1213, 2013, Lohse et al. Cancer Res 76:403, 2016). Here, we compared the efficiency of IgG1 and IgA2 isotype variants of the type I CD20 antibody 1F5 in killing lymphoma cells in vitro and in vivo.Methods: Recombinant antibodies against human CD20 were produced by co-transfecting BHK cells with vectors encoding the 1F5 variable, Igα2 or Igγ1 heavy, and κ light chain constant regions, respectively. The resulting isotype variants were compared for their biochemical characteristics as well as Fab- and Fc- mediated effector functions using human lymphoma cell lines as targets. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice xenotransplanted with human RAJI lymphoma cells were employed to investigate the therapeutic efficacy of human CD20 antibodies. Additionally, in vivo depletion of human CD20 transgenic B cells was evaluated in a syngeneic B cell depletion model using wildtype, human FcαRI transgenic and C3 knock-out mice.Results and Discussion:In vivo studies with xenotransplanted RAJI cells in NSG mice, which lack functional T and NK cells, demonstrated significantly prolonged survival of treated as compared to non-treated mice, indicating that myeloid effector cells may contribute to the therapeutic efficacy of CD20 antibodies against human lymphoma cells. In vitro, neither IgG1 nor IgA2 variants of 1F5 showed efficient Fab-mediated effects such as direct cell death induction and homotypic aggregation compared to type II antibodies. However, human IgA2 but not IgG1 antibody variants against CD20 effectively triggered ADCC by human PMN, the most numerous myeloid effector cell population. Although IgA does not bind C1q, CD20 IgA antibodies also triggered CDC against several lymphoma cell lines. CDC was predominantly mediated by the alternative pathway, as evidenced by the kinetics of lysis, the requirement for higher serum concentrations and inhibition by the C3 inhibitor compstatin. Further in vivo experiments demonstrated that 1F5-IgA2 effectively depleted B cells in a syngeneic human CD20 transgenic B cell depletion model. However, studies in human FcαRI transgenic or C3 knock-out mice indicated that B cell depletion was not mediated by FcαRI or complement - suggesting that other currently undefined mechanisms contribute to the in vivo efficacy of IgA antibodies against CD20.Conclusions: Together, the presented results suggest that CD20 antibodies of human IgA isotype constitute promising immunotherapeutic reagents with unique effector functions. Additional studies are required to further elucidate their effector mechanisms in vitro and in vivo. DisclosuresCragg:Roche: Consultancy, Research Funding; Gilead Sciences: Research Funding; Baxalta: Consultancy; Bioinvent International: Consultancy, Research Funding; GSK: Research Funding.

Abstract 1488: Development of anti-nucleolin antibodies with broad spectrum anticancer activity and negligible toxicity to normal cells

Abstract Nucleolin has multiple, unique functions in cancer cells, including the shuttling of ligands from the cell surface to the cytoplasm and the stabilization of oncogene and cytokine mRNAs that have an AU-rich nucleolin binding element in their 3’-UTRs. We developed a panel of anti-nucleolin antibodies that exploit the temperature-dependent shuttling function of nucleolin to gain access to the cytoplasm of human tumor cells and induce oncogene mRNA destabilization. Several lines of evidence indicate that our fully human monoclonal IgG1 antibody, CP101.2C8, targets nucleolin and penetrates tumor cells. CP101.2C8 bound tightly to human recombinant nucleolin (Kd = 26 ± 7 nM, SEM.) and to plasma membrane nucleolin of human tumor cells. Confocal microscopy of Panc-1 and DU-145 tumor cells incubated at 37 0C with CP101.2C8 revealed punctate localization of the antibody in the plasma membranes of these cells and internalization of the antibody into the cytoplasm. The localization of the antibody within foci in the plasma membrane suggested that the antibody was bound to nucleolin that was incorporated into lipid rafts within the plasma membrane. MCF-7 human breast cancer cells were made more than 100-fold resistant to CP101.2C8 by growing the cells in increasing concentrations of the antibody. The resistant cells regained contact inhibition and had a 10-fold lower level of cytoplasmic nucleolin compared to the parental MCF-7 cells. CP101.2C8 is a potent inhibitor of tumor cell viability in vitro. IC50 values of less than 1 μg/ml were obtained for CP101.2C8 versus MV4-11 AML cells, colon, prostate, and lung cancer cells as well as CD33+-CD24- stem cells from MDA-MD-231 breast cancer cells. In contrast, the IC50 concentrations of CP101.2C8 versus normal human B and myeloid cells, breast epithelial cells and lung fibroblasts were greater than 10 μg/ml. Unlike the tumor cells, these normal cells did not express detectable levels of nucleolin in either the plasma membrane or cytoplasm. A fully human recombinant CP101.2C8 is also potent in reducing the viability of MV4-11 cells (IC50 = 0.4 μg/ml). Groups of 10 nu/nu mice bearing SC MV4-11 tumor xenografts were injected IV (every 3 days x 6) with either 10 mg/kg CP101.2C8 or 10 mg/kg of a human IgG1 isotype control antibody. Tumor progression to the 2,000 mm3 endpoint was observed between 24-41 days in all mice treated with the control antibody, while 3/10 of the CP101.2C8-treated mice were long-term survivors that did not reach the endpoint by day 76. The hazard ratio calculated from the Kaplan-Meier plot was 0.22. CP101.2C8 was well tolerated by all the mice; the only adverse event observed was a transient loss in mean body weight of 16%. The widespread and aberrant expression of the multi-functional protein nucleolin in human tumor cells, in contrast to the corresponding normal cells, explains both the broad-spectrum anticancer activity and tumor selectivity of antibody CP101.2C8. Citation Format: Daniel J. Fernandes, Baby G. Tholanikunnel. Development of anti-nucleolin antibodies with broad spectrum anticancer activity and negligible toxicity to normal cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1488.

Abstract B179: Enhancing intratumoural Treg depletion through antibody engineering and analysis of the checkpoint landscape of mouse and human cancers

Abstract Different components of the tumor microenvironment hamper the immune response against cancer cells and are a critical impediment for the success of cancer immunotherapy. Among these, the accumulation of regulatory T cells (Treg) and the reduction of the effector T cells (Teff)/Treg ratio favors tumor progression and is associated with worse prognosis in several human cancers. Treg elimination in the tumor microenvironment can be achieved through antibodies that target molecules expressed preferentially in Treg and that induce antibody-mediated cell cytotoxicity (ADCC). CTLA-4 is highly expressed on tumor-infiltrating Treg and therapy with anti-CTLA-4 has been effective for cancer treatment in murine models and in humans. In mice, we and others have shown that anti-CTLA-4 depletes Treg in the tumor but not in peripheral lymphoid organs. This site specificity in mice is given by the expression of the Fcγ-receptor IV (FcγRIV) in myeloid cells present in the tumor microenvironment but not in the periphery. However, the role of FcγRs in the effectiveness of anti-CTLA-4 therapy or therapies with other immunomodulatory antibodies in humans is not known. We therefore investigated the density of targets of clinically relevant immunomodulatory antibodies in different subpopulations of tumor-infiltrating immune cells as well as the expression of FcγR subtypes in murine tumor models. Because of the inter-species differences in the repertoire and the cellular distribution of FcγRs, we also studied this in humanized-FcγR (huFcγR) mice, in which there is no expression of murine FcγRs but of their human homologues. We compared this with samples of human melanoma and observed surprisingly similar expression profiles of both FcγRs and targets of immunomodulatory antibodies, including CTLA-4. We then asked whether anti-CTLA-4 would also lead to selective Treg depletion in the tumor microenvironment in huFcγR mice. Using an antibody targeting mCTLA-4 with a human IgG1 isotype, we observed that was the case. Furthermore, mutation of the Fc region of the antibody to enhance its affinity to activating huFcγRs resulted in more effective selective Treg depletion in the tumor. These findings are not only relevant for CTLA-4 but also for other targets expressed preferentially on Treg instead of Teff and must be taken into account when designing immunomodulatory antibodies for clinical use. Citation Format: Frederick Arce Vargas, Andrew J. Furness, Marta H. Lesko, Martin Pule, Jeffrey V. Ravetch, Karl S. Peggs, Sergio A. Quezada. Enhancing intratumoural Treg depletion through antibody engineering and analysis of the checkpoint landscape of mouse and human cancers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B179.

Abstract A054: In vivo characterization of Ipilimumab T cell modulation and antitumor activity in a tumor bearing humanized NSG mouse model

Abstract The purpose of these studies was to determine the effect of Ipilimumab treatment on T cell expansion, activation, cytokine production, tumor infiltration, and expression level of co-stimulatory and co-inhibitory receptors in a tumor bearing humanized NSG mouse model. Antibody therapies for immune modulation are proving to be effective in the oncology setting, as demonstrated by anti-CTLA-4 Ipilimumab and anti-PD-1 Pembrolizumab clinical activity. In order to assess efficacy of new therapies in the context of immune activation and tumor response, there is a need for suitable preclinical in vivo models. One approach to study immune cell function is to inject human peripheral blood mononuclear cells (PBMCs) into adult immunodeficient NSG (NOD/SCID/IL-2Rγnull) mice. This model, known as Hu-PBMC NSG, induces a Graft-versus-Host Disease (GvHD) state and has been used to study effector and memory T cell activity. Here we utilized the Hu-PBMC NSG model implanted with human cancer cell lines to investigate the effect of Ipilimumab on T cells and tumor growth in vivo. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee at GSK. The human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents. We confirmed human PBMC engraftment in NSG mice, validated tumor growth of human A2058 melanoma and 786-O renal adenocarcinoma cell lines, and dosed Ipilimumab in tumor bearing NSG mice with different human PBMC donors. Naïve NSG mice were intravenously injected with 20x106 human PBMCs and the kinetics of human cell engraftment was monitored weekly. For studies including Ipilimumab dosing, mice were inoculated with a subcutaneous injection of 2.5x106 A2058 or 1x106 786-O tumor cells. As tumor size reached 100 mm3, mice were randomized and injected with 20x106 human PBMCs. Two days later, mice were intraperitoneally treated with Ipilimumab or human IgG1 isotype control twice weekly for 6 doses total. Tumor growth and body weight was evaluated over time. Peripheral blood was collected weekly for analysis of T cell activation, receptor expression levels, and human cytokine production until mice developed GvHD or tumor volume reached 2,000 mm3. Select tumors were also harvested for tumor infiltrating lymphocyte (TIL) analysis by flow cytometry. Our studies showed NSG mice demonstrated similar human CD45+ cell engraftment in blood with various PBMC donors. The frequency of circulating human CD45+ lymphocytes in mouse peripheral blood increased to 50% or greater until week 4. 95% of the CD45+ cells were CD3+ T cells, containing both CD4+ and CD8+ subsets. Signs of GvHD were observed at week 4, and serum cytokine analysis showed high levels of GvHD markers e.g. IL5, IL10, and TNF-alpha. Ipilimumab treatment delayed tumor growth, increased the expansion of human CD45+ cells, and induced higher levels of TNF-alpha, IL-12p70, IL-13, and IL-5 cytokines compared to isotype control. Ipilimumab also increased the surface expression level of CD69, PD-1, OX40, ICOS, CD137, TIM3, and LAG3 on circulating T cells, and increased the number of A2058 tumor infiltrating lymphocytes. TIL analysis showed that compared to isotype control, Ipilimumab increased expression of CD69, PD-1, OX40, and ICOS on tumor infiltrating lymphocytes. In conclusion, Ipilimumab treatment increased T cell expansion, activation, expression of co-stimulatory and co-inhibitory receptors, and cytokine production in this tumor-bearing Hu-PBMC NSG model. It also increased the number of tumor infiltrating lymphocytes with a corresponding tumor growth delay. Based on Ipilimumab activity, this model can be utilized to assess pre-clinical efficacy of novel immunotherapies. Citation Format: Meixia Bi, Chris Hopson, Tianqian Zhang, James Smothers, Axel Hoos. In vivo characterization of Ipilimumab T cell modulation and antitumor activity in a tumor bearing humanized NSG mouse model. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A054.

A peptide mimic blocks the cross-reaction of anti-DNA antibodies with glomerular antigens

Anti-DNA antibodies play a pivotal role in the pathogenesis of lupus nephritis by cross-reacting with renal antigens. Previously, we demonstrated that the binding affinity of anti-DNA antibodies to self-antigens is isotype-dependent. Furthermore, significant variability in renal pathogenicity was seen among a panel of anti-DNA isotypes [derived from a single murine immunoglobulin (Ig)G3 monoclonal antibody, PL9-11] that share identical variable regions. In this study, we sought to select peptide mimics that effectively inhibit the binding of all murine and human anti-DNA IgG isotypes to glomerular antigens. The PL9-11 panel of IgG anti-DNA antibodies (IgG1, IgG2a, IgG2b and IgG3) was used for screening a 12-mer phage display library. Binding affinity was determined by surface plasmon resonance. Enzyme-linked immunosorbent assay (ELISA), flow cytometry and glomerular binding assays were used for the assessment of peptide inhibition of antibody binding to nuclear and kidney antigens. We identified a 12 amino acid peptide (ALWPPNLHAWVP, or 'ALW') which binds to all PL9-11 IgG isotypes. Preincubation with the ALW peptide reduced the binding of the PL9-11 anti-DNA antibodies to DNA, laminin, mesangial cells and isolated glomeruli significantly. Furthermore, we confirmed the specificity of the amino acid sequence in the binding of ALW to anti-DNA antibodies by alanine scanning. Finally, ALW inhibited the binding of murine and human lupus sera to dsDNA and glomeruli significantly. In conclusion, by inhibiting the binding of polyclonal anti-DNA antibodies to autoantigens in vivo, the ALW peptide (or its derivatives) may potentially be a useful approach to block anti-DNA antibody binding to renal tissue.

Expression Profile of CD38 and Related Ectoenzymes in Myeloma Bone Niche: A Rational Basis for the Use of Daratumumab to Inhibit Osteoclast Formation and Activity

The relationship between multiple myeloma (MM) cells and osteoblast (OB)s and osteoclast (OCL)s into the bone marrow (BM) niche has a critical role in the pathophysiology of MM and in the development of bone disease in MM patients.Recently, Daratumumab (DARA), a human anti-CD38 monoclonal antibody has been developed with broad-spectrum killing activity including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and, at least in part, modulation of CD38 enzymatic activity. In pre-clinical studies, DARA has been shown to effectively kill MM cells and clinical trials are ongoing. However, the effects of DARA in the context of the MM bone niche and on MM-induced bone remodeling alterations are unknown.In this study, we have firstly evaluated the expression profile of CD38 and its related ectoenzymes by bone niche cells and, thereafter, we investigated the effect of DARA on OCL and OB formation and bone remodeling. The study design included immunohistochemistry for CD38, CD73, CD39, CD203a (PC-1), CD157 and CD31 on bone biopsies in a cohort of 38 patients with newly diagnosed MM and 14 patients with monoclonal gammopathy of uncertain significance (MGUS). Moreover, the expression profile of these antigens was performed by flow cytometry on primary BM CD138+ cells (sample number=16), mesenchymal stromal cells, OBs, monocytes and OCLs and on the related human cell lines.As expected, we found that CD38 was highly expressed by MM cells that were also positive for CD203a and for CD39 and CD31 at variable level but not for CD157 and CD73. CD38 was also expressed by BM monocytes but not by OBs, OCLs and BM stromal cells. Interestingly, we found that OBs expressed CD73 and CD203a. Any significant difference was not observed in the expression of CD38 and related ectoenzymes between MM and MGUS patients. In line with CD38 expression profile by MM cells and the niche, we further demonstrated that DARA binds both MM cells and monocytes, but not OBs and OCLs. Consistently, we lack to find any significant effect of DARA on OB formation from BM stromal cells or OBs proliferation and survival. Thus, we investigated the effect of DARA (1-25 ug/ml) as compared to human IgG isotype control on OCL formation and activity in the presence of RANKL and M-CSF, using either CD138- cell fraction or purified CD14+ cells from MM BM samples. OCLs were evaluated by both TRAP staining and a fluorimetric osteolysis assay. We found that DARA, between 10 and 25 ug/ml, with a dose dependent effect, significantly inhibited OCL formation and activity from BM mononuclear cells and from the CD138- cell fraction, but not from purified CD14+ cells. The inhibitory effect on OCL formation by DARA was observed when the antibody was present for all the culture period (21-28 days). On the other hand DARA did not show any effect on late OCL progenitors and mature OCLs. Accordingly CD38 expression by monocytes cultured in a pro-osteoclastogenic medium disappeared after 7 days.In conclusion, our data indicate that DARA inhibit osteoclastogenesis, likely mediated by ADCC, targeting monocytes and early OCL progenitors. This evidence supports the use of an anti-CD38 based approach as a treatment for MM bone disease. DisclosuresMalavasi:Janssen: Honoraria, Research Funding. Giuliani:Celgene Italy: Other: Research Grant; Janssen Pharamceutical: Other: Research Grant.

Targeting Mechanisms for Natural Killer Cell Dysfunction in Patients with Multiple Myeloma

Patients with multiple myeloma (MM) have a multi-faceted immune dysfunction with poor NK cell cytotoxic function. We show that NK cells in MM have a disturbed homeostasis, featured by an increased frequency of terminally differentiated CD57+ NK cells and almost complete absence of the less mature CD56hi NK cell subset. MM patient NK cells have reduced levels of killer activating receptors (KAR) [Nkp46, Nkp44 and NKG2D] and increased levels of killer inhibitory receptor (KIR) [KIR2DL1 and KIR2DL3]. Despite expressing normal levels of the NK effector transcription factors Tbet and eomesdermin, and the effector protein perforin, MM patient NK cells demonstrate poor cytotoxic function against myeloma cell lines and lower levels of the cytotoxic proteins granzyme B (gzmB).Elotuzumab (Elo, Bristol Myers Squibb) is a human IgG1 antibody which targets SLAM-F7, a protein highly expressed on myeloma plasma cells and on specific subsets of immune cells. Elo mediates dual activation of NK cells and has shown promising clinical responses when combined with lenalidomide; however the complete mechanism(s) whereby Elo mediates these effects have not been fully described. Thus, this project investigates the Elo mechanisms of action in myeloma. To explore whether MM patient NK cells were capable of ADCC, we examined NK cell subset degranulation (CD107a) and IFN-γ secretion when co-cultured with the SLAM-F7 expressing OPM-2 myeloma cells and Elo. Healthy donor NK cells degranulated in response to Elo-labeled OPM-2 cells, which correlated with loss of the CD56lo CD16+ NK cell subset. In contrast, newly diagnosed (NEW) and refractory/relapsed MM (RRMM) patient NK cells had a decreased response to Elo-labeled OPM-2 cells as the CD56lo CD16+ NKsubset was retained, and they degranulated at a significantly lower level than that observed in healthy donor NK cells. Therefore, all MM patient NK subsets (CD56lo CD16+, CD161+ and CD57+) assessed were low responders to Elo-labeled OPM-2 cells.We then visually examined NK cell killing kinetics of Elo-labeled myeloma target cells using time lapse live video microscopy (TLLVM). NK cells used ADCC to kill OPM-2 cells in the presence of Elo, the killing kinetics of was equivalent to direct killing induced in presence of the human IgG isotype control, the delivery of the lethal hit occurring in under three minutes. Elo also mediated serial killing of OPM-2 cells. Interestingly, Elo did delay the NK cell detachment from dying myeloma plasma cell targets, which results in prolonged signaling to the NK cell and consequently higher levels of secreted cytokines and pro-inflammatory chemokines. Taken together, this data indicates Elo-induces a profound change in the NK-mediated killing kinetics of myeloma cells and that likely recruits other immune effectors to the myeloma killing site.Given that NK cells can also express SLAM-F7, we assessed whether they were likely to be a target for other NK cells when bound to Elo, and induce fratricide. We used killing assays, flow cytometry and TLLVM to show that in the presence of Elo, healthy donor NK cells were capable of NK fratricide under the assay conditions tested. Interestingly, fratricide induced death saw the depletion of the CD56lo CD16+ NKsubset. This data may partially explain why there is a rapid but transient reduction of NK cells from the peripheral blood of MM patients who received single agent Elo.In summary, MM patient NK dysfunction is due to aberrant NK cell homeostasis featured by terminal differentiation of the NK cells to a subset with poor natural cytotoxic function and high KIR expression. In vitro, Elo is less effective at inducing ADCC responses by NK cells from MM patients compared to normal donors. Nonetheless, Elo induces efficient normal donor NK cell killing of myeloma target cells, the killing kinetics is featured by a protracted immune synapse time and secretion of high levels of pro-inflammatory cytokines/chemokines by 24 hours, plus serial killing of myeloma target cells. These data likely explain the single agent efficacy of Elo in RRMM patients. The data also underlines the exciting potential of Elo to induce NK mediated ADCC of myeloma plasma cells, and recruit multiple arms of the immune system to the killing site. To harness its full potential, combination of Elo with other drugs (eg. Lenalidomide and anti-KIR) maybe required to normalize MM patient NK cell function and initiate the immune attack. DisclosuresRobbins:Bristol-Myers Squibb: Employment, Other: shareholder. Neeson:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Accumulation of secreted antibodies in plant cell cultures varies according to the isotype, host species and culture conditions

Nicotiana tabacum suspension cells have been widely used to produce monoclonal antibodies, but the yield of secreted antibodies is usually low probably because of proteolytic degradation. Most IgGs that have been expressed in suspension cells have been of the human IgG1 isotype. In this study, we examined whether other isotypes displayed the same sensitivity to proteolytic degradation and whether the choice of plant host species mattered. Human serum IgG displayed different degradation profiles when incubated in spent culture medium from N. tabacum, Nicotiana benthamiana or Arabidopsis thaliana suspension cells. Zymography showed that the protease profile was host species dependent. Three human isotypes, IgG1, IgG2 and IgG4, and a mouse IgG2a were provided with the same heavy- and light-chain variable regions from an anti-human IgM antibody and expressed in N. tabacum cv. BY-2 and A. thaliana cv. Col-0 cells. Although all tested isotypes were detected in the extracellular medium using SDS-PAGE and a functional ELISA, up to 10-fold differences in the level of intact antibody were found according to the isotype expressed, to the host species and to the culture conditions. In the best combination (BY-2 cells secreting human IgG1), we reported accumulation of more than 30 mg/L of intact antibody in culture medium. The possibility of using IgG constant regions as a scaffold to allow stable accumulation of antibodies with different variable regions was demonstrated for human IgG2 and mouse IgG2a.

Physiological Level Production of Antigen-Specific Human Immunoglobulin in Cloned Transchromosomic Cattle

Therapeutic human polyclonal antibodies (hpAbs) derived from pooled plasma from human donors are Food and Drug Administration approved biologics used in the treatment of a variety of human diseases. Powered by the natural diversity of immune response, hpAbs are effective in treating diseases caused by complex or quickly-evolving antigens such as viruses. We previously showed that transchromosomic (Tc) cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin heavy-chain (hIGH) and kappa-chain (hIGK) germline loci (named as κHAC) are capable of producing functional hpAbs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, are homozygously inactivated (double knockouts or DKO). However, B lymphocyte development in these Tc cattle is compromised, and the overall production of hpAbs is low. Here, we report the construction of an improved HAC, designated as cKSL-HACΔ, by incorporating all of the human immunoglobulin germline loci into the HAC. Furthermore, for avoiding the possible human-bovine interspecies incompatibility between the human immunoglobulin mu chain protein (hIgM) and bovine transmembrane α and β immunoglobulins (bIgα and bIgβ) in the pre-B cell receptor (pre-BCR) complex, we partially replaced (bovinized) the hIgM constant domain with the counterpart of bovine IgM (bIgM) that is involved in the interaction between bIgM and bIgα/Igβ; human IgM bovinization would also improve the functionality of hIgM in supporting B cell activation and proliferation. We also report the successful production of DKO Tc cattle carrying the cKSL-HACΔ (cKSL-HACΔ/DKO), the dramatic improvement of B cell development in these cattle and the high level production of hpAbs (as measured for the human IgG isotype) in the plasma. We further demonstrate that, upon immunization by tumor immunogens, high titer tumor immunogen-specific human IgG (hIgG) can be produced from such Tc cattle.

FcγRΙΙB controls the potency of agonistic anti-TNFR mAbs.

Isotype plays a crucial role in therapeutic monoclonal antibody (mAb) function, mediated in large part through differences in Fcγ receptor (FcγR) interaction. Monoclonal Abs such as rituximab and alemtuzumab, which bind target cells directly, are designed for efficient recruitment of immune effector cells through their activatory FcγR engagement to mediate maximal target cell killing. In this setting, binding to inhibitory FcγRIIB is thought to inhibit function, making mAbs with high activatory/inhibitory (A/I) FcγR binding ratios, such as mouse IgG2a and human IgG1, the first choice for this role. In contrast, exciting new data show that agonistic mAbs directed against the tumour necrosis factor receptor superfamily member CD40 require interaction with FcγRIIB for in vivo function. Such ligation activates antigen-presenting cells, promotes myeloid and CTL responses and potentially stimulates effective anti-cancer immunity. It appears that the role of FcγRIIB is to mediate mAb hyper-crosslinking to allow CD40 downstream intracellular signalling. Previous work has shown that mAbs directed against other TNFR family members, Fas and death receptor 5 and probably death receptor 4, also require FcγRIIB hyper-crosslinking to promote target cell apoptosis, suggesting a common mechanism of action. In mouse models, IgG1 is optimal for these agents as it binds to FcγRIIB with tenfold higher affinity than IgG2a and hence has a relatively low A:I FcγR binding ratio. In contrast, human IgG isotypes have a universally low affinity for FcγRIIB, but in the case of human IgG1, engineering the Fc to increase its affinity for FcγRIIB can potentially overcome this problem. Thus, modifying the A/I binding ratio of human IgG Fc can be used to optimise different types of therapeutic activity by enhancing cytotoxic or hyper-crosslinking function.

Crosstalk between Human IgG Isotypes and Murine Effector Cells

Development of human therapeutic Abs has led to reduced immunogenicity and optimal interactions with the human immune system in patients. Humanization had as a consequence that efficacy studies performed in mouse models, which represent a crucial step in preclinical development, are more difficult to interpret because of gaps in our knowledge of the activation of murine effector cells by human IgG (hIgG) remain. We therefore developed full sets of human and mouse isotype variants of human Abs targeting epidermal growth factor receptor and CD20 to explore the crosstalk with mouse FcγRs (mFcγRs) and murine effector cells. Analysis of mFcγR binding demonstrated that hIgG1 and hIgG3 bound to all four mFcγRs, with hIgG3 having the highest affinity. hIgG1 nevertheless was more potent than hIgG3 in inducing Ab-dependent cellular cytotoxicity (ADCC) and Ab-dependent cellular phagocytosis with mouse NK cells, mouse polymorphonuclear leukocytes, and mouse macrophages. hIgG4 bound to all mFcγRs except mFcγRIV and showed comparable interactions with murine effector cells to hIgG3. hIgG4 is thus active in the murine immune system, in contrast with its inert phenotype in the human system. hIgG2 bound to mFcγRIIb and mFcγRIII, and induced potent ADCC with mouse NK cells and mouse polymorphonuclear leukocytes. hIgG2 induced weak ADCC and, remarkably, was unable to induce Ab-dependent cellular phagocytosis with mouse macrophages. Finally, the isotypes were studied in s.c. and i.v. tumor xenograft models, which confirmed hIgG1 to be the most potent human isotype in mouse models. These data enhance our understanding of the crosstalk between hIgGs and murine effector cells, permitting a better interpretation of human Ab efficacy studies in mouse models.

Inhibitory human antichimeric antibodies to rituximab in a patient with pemphigus

Inhibitory human antichimeric antibodies to rituximab in a patient with pemphigus

Development of a Human Anti-CD27 Antibody with Efficacy in Lymphoma and Leukemia Models by Two Distinct Mechanisms

Abstract 2861CD27, a lymphoid cell-specific TNFR superfamily member, is constitutively expressed on the majority of T cells, some NK cells and memory B cells. Through interaction with its ligand CD70, CD27 transduces a co-stimulatory signal promoting T cell and NK cell activation and cytotoxicity. In addition, CD27 is also expressed on many lymphoid-originated hematological neoplastic cells, such as chronic lymphocytic leukemia, mantle cell lymphoma, and Waldenstrom macroglobulinemia, thus being a potential direct target for antibody therapy. To generate potential antibodies for clinical development, we immunized human Ig transgenic mice and developed a panel of CD27 specific human mAbs. Clone 1F5 was identified as a lead based on its high affinity to both human and monkey CD27, enhanced co-stimulation of T cells, and ADCC of CD27-expressing lymphoblastic cell lines.Using SCID mice challenged with CD27-expressing human lymphoid cell lines, we demonstrated that 1F5 mediates conventional antibody effector function. Compared to human IgG1 isotype control (huIgG1), 1F5 at doses ranging 33 μg – 500 μg (x 6) significantly delayed the growth of Burkitt’s lymphoma Raji even when administration was initiated 1 week after tumor inoculation. Similar anti-tumor activity was observed against other CD27-expressing tumor lines including, Daudi and T-originated acute lymphoblastic leukemia CCRF-CEM.In order to investigate 1F5 in vivo agonistic activities and T cell-mediated tumor eradication, a human CD27 transgenic mouse model (hCD27-Tg) was generated and backcrossed onto C57BL/6 and BALB/c backgrounds. The expression profile and regulation of the human CD27 transgene driven by its own promoter were similar to that observed with endogenous mouse CD27. In addition to enhancing T cell responses when combined with vaccination, 1F5 treatment was highly effective against syngeneic mouse tumors including lymphoma BCL1 (BALB/c) and thymoma EL4-derived E.G7 (C57BL/6). For the BCL1 model, various dose levels of 1F5 mAb were delivered to mice intraperitoneally on days 3, 5, 7, 9 and 11 after i.v. administration of 107 BCL1 cells to huCD27 Tg and control animals. Controls including hCD27-Tg mice treated with saline or isotype control, or WT mice treated with 1F5 all performed consistently, leading to 50% survival approximately 23 days after tumor challenge. Treatment of mice with mAb, 1F5 substantially improved the 50% survival in a dose dependent fashion to >70 days post tumor challenge at the higher dose levels.Based on the promising efficacy data with anti-CD27 mAb 1F5 in immunocompromised and immunocompetent lymphoma models, a clinical grade product, referred to as CDX-1127 was manufactured and tested for safety. To assess the potential for 1F5 to mediate lymphocyte activation, we investigated its ability to induce proliferation and cytokine release from human PBMC or purified T cell cultures. Consistent with the known biology of CD27 we demonstrated the 1F5 mAb does not lead to direct activation of lymphocytes in the absence of additional signals. However, combining 1F5 with suboptimal levels of T cell receptor stimulation using anti-CD3 mAb (OKT3) was shown to enhance proliferation of human T cells. Two studies were performed using cynomolgus macaques. There were no CDX-1127 related mortalities or changes noted in the clinical condition, food appetence, body weights and body temperature, ophthalmic, electrocardiographic and clinical pathology assessments, organ weights and bone marrow assessments. In addition, there were no major differences in the percentage of lymphocyte populations between control and CDX-1127 treated animals at the end of the study demonstrating that the antibody did not significantly deplete normal CD27-expresssing cells.Based on the pre-clinical studies we are planning a Phase 1 clinical trial of CDX-1127 in patients with hematological malignancies and selected solid tumors. The trial is designed with separate arms to independently assess the safety and activity of CDX-1127 in hematologic malignancies, in which the antibody may act through multiple mechanisms, and in solid tumors where it would be fully dependent on indirect immune mechanisms. Disclosures:He:Celldex Therapeutics, Inc.: Employment. Thomas:Celldex Therapeutics, Inc.: Employment. Weidlick:Celldex Therapeutics, Inc.: Employment. Vitale:Celldex Therapeutics, Inc.: Employment. O'Neill:Celldex Therapeutics, Inc.: Employment. Prostak:Celldex Therapeutics, Inc.: Employment. Sundarapandiyan:Celldex Therapeutics, Inc.: Employment. Marsh:Celldex Therapeutics, Inc.: Employment. Yellin:Celldex Therapeutics, Inc.: Employment. Davis:Celldex Therapeutics, Inc.: Employment. Keler:Celldex Therapeutics, Inc.: Employment.

The in vitro resistance of IgG2 to proteolytic attack concurs with a comparative paucity of autoantibodies against peptide analogs of the IgG2 hinge

The mammalian antibody repertoire comprises immunoglobulin (Ig) molecules of multiple isotypes and subclasses with varying functional properties. Among the four subclasses of the human IgG isotype, we found that IgG2 exhibits a particular resistance to human and bacterial proteases that readily cleave the IgG1 hinge region in vitro. Autoantibodies (IgGs) that recognize points of proteolytic cleavage in the IgG1 hinge are widespread in the healthy human population, suggesting that IgG1 fragmentation and the generation of cryptic antigens for host immune surveillance commonly occur in vivo. We previously reported that autoantibodies to cleaved IgG1s can restore Fc-mediated effector functions that are lost following proteolytic cleavage of the hinge. In contrast, it was not possible to demonstrate an analogous cohort of autoantibodies to IgG2 hinge epitope analogs, and there appeared to be no functional component in human serum with the ability to reconstitute Fc effector functions to a cell-bound IgG2 fragment. Thus, the results indicate that among the IgG subclasses, human IgG2 is uniquely resistant to a number of known pathological proteases and that autoimmune recognition to potential cleavage points in the IgG2 hinge appears to be absent in human circulation.

Human IgG isotypes and activating Fcγ receptors in the interaction of Salmonella enterica serovar Typhimurium with phagocytic cells.

Several classes and multiple subclasses of immunoglobulins are produced towards protein and polysaccharide antigens in response to Salmonella infection and play a key role in protection against systemic disease. The targeting of Salmonella to Fc receptors (FcR) on phagocytes is a key step in the antibody-mediated antibacterial functions of host cells. We wished to compare the relative efficiency of different human IgG subclasses, which targeted the Salmonella enterica OmpA surface protein in modulating the interaction of bacteria with human phagocytes. To this end, we developed a novel system by tagging OmpA with a foreign CD52 mimotope (TSSPSAD) and opsonizing the bacteria with a panel of humanized CD52 antibodies that share the same antigen-binding V-region, but have constant regions of different subclasses. Our data revealed that opsonization with all the IgG subclasses increases Salmonella uptake by human phagocytes. IgG3 resulted in the highest level of bacterial uptake and the highest average bacterial load per infected cell, which was closely followed by IgG1, then IgG4 and lastly IgG2. Phagocytosis mediated by IgG1, IgG3 and IgG4 had a higher dependency on FcγRI than FcγRIIA, whereas IgG2-mediated phagocytosis required FcγRIIA more than FcγRI. The results show that IgG binding to OmpA increases the uptake of Salmonella by human phagocytic cells and that the efficiency of this process depends both on the subclass of the IgG and the type of FcR that is available for antibody binding.

Impact of Human IgA Antibodies on Complement-Dependent Cytotoxicity Mediated by Combinations of EGF-R-Directed Antibodies

Dual combinations of non-crossblocking epidermal growth factor receptor (EGF-R)-directed monoclonal antibodies were demonstrated to effectively induce complement-dependent cytotoxicity (CDC) of tumor cells, whereas individual antibodies were ineffective. Here the modulating effects of different antibody isotypes on CDC were studied by adding them as a third antibody. Two different combinations of non-crossblocking EGF-R antibodies of human IgG1 isotype, 018/003 and 425/005, were investigated against the A431 and A1207 cell lines. As a third antibody, human IgG1, IgA1, and IgA2 isotype variants of the therapeutic EGF-R antibody 225 were employed that bind to an EGF-R epitope distinct from the other EGF-R antibodies. In this model, the human IgG1 antibody proved to further enhance CDC, whereas both IgA antibodies significantly blocked CDC. The IgG1 and IgA variants increased target opsonization at similar levels, but the isotypes differed in their effects on C1q fixation. Addition of IgG1 significantly enhanced complement factor binding on the target surface, whereas both IgA antibodies reduced complement binding. Control experiments revealed this blocking effect to be not specific to IgA antibodies, but to antibody constructs incapable of activating the complement system. Interestingly, the effects caused by the IgA2 isotype were consistently stronger than those by IgA1, which may be caused by stronger steric hindrance due to its reduced hinge flexibility. These results demonstrate that monoclonal IgA antibodies inhibit IgG-mediated complement activation in vitro and suggest that the appearance of IgA antibodies within a polyclonal immune response might inhibit complement activation in vivo.

Abstract 5343: Development of novel anti-CD27 human antibodies with therapeutic potential

Abstract The costimulatory molecule CD27 is a member of the tumor necrosis factor (TNF) receptor superfamily, and is constitutively expressed on the majority of mature T cells, memory B cells, and a portion of NK cells. The interaction of CD27 with its ligand CD70 plays key roles in T cell activation, proliferation, survival, maturation of effector capacity and memory; in clonal B cell expansion and germinal center formation; and in NK cell cytolytic activity. Previous reports have shown that antigen-specific CD8+ cytolytic T cell immunity can be augmented through CD27 activation in mice. Of particular relevance is the work by M. J. Glennie et al. demonstrating that an agonistic anti-mouse CD27 mAb given without a DC maturation signal has potent anti-tumor activity through boosting of T cell immunity. To explore the therapeutic potential of this target, a panel of fully human antibodies recognizing human CD27 was generated using human Ig transgenic mice immunized with recombinant human CD27. These human anti-CD27 mAbs showed specific and high affinity binding to CD27 and CD27-expressing lymphoma cells. We generated transgenic mice that express human CD27 (hCD27-Tg) to evaluate the anti-human CD27 mAbs in vivo. The expression profile and regulation of the human CD27 transgene driven by its own promoter were similar to that observed with endogenous mouse CD27. Administration of anti-human CD27 mAbs in combination with ovalbumin greatly enhanced antigen specific T cell responses compared to ovalbumin plus human IgG1 isotype control in the hCD27-Tg mice. The SIINFEKL-specific CD8+ T cell proliferation and activation were detected by pentamer staining and IFNγ ELISPOT analysis. These data support the further development of these novel human anti-CD27 antibodies and we are currently investigating their therapeutic activity using various in vivo tumor models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5343.

Human IgG2 Antibodies against Epidermal Growth Factor Receptor Effectively Trigger Antibody-Dependent Cellular Cytotoxicity but, in Contrast to IgG1, Only by Cells of Myeloid Lineage

Ab-dependent cellular cytotoxicity (ADCC) is usually considered an important mechanism of action for immunotherapy with human IgG1 but not IgG2 Abs. The epidermal growth factor receptor (EGF-R) Ab panitumumab represents the only human IgG2 Ab approved for immunotherapy and inhibition of EGF-R signaling has been described as its principal mechanism of action. In this study, we investigated effector mechanisms of panitumumab compared with zalutumumab, an EGF-R Ab of the human IgG1 isotype. Notably, panitumumab was as effective as zalutumumab in recruiting ADCC by myeloid effector cells (i.e., neutrophils and monocytes) in contrast to NK cell-mediated ADCC, which was only induced by the IgG1 Ab. Neutrophil-mediated tumor cell killing could be stimulated by myeloid growth factors and was triggered via FcgammaRIIa. Panitumumab-mediated ADCC was significantly affected by the functional FcgammaRIIa-R131H polymorphism and was induced more effectively by neutrophils from FcgammaRIIa-131H homozygous donors than from -131R individuals. This polymorphism did not affect neutrophil ADCC induced by the IgG1 Ab zalutumumab. The in vivo activity of both Abs was assessed in two animal models: a high-dose model, in which signaling inhibition is a dominant mechanism of action, and a low-dose model, in which effector cell recruitment plays a prominent role. Zalutumumab was more effective than panitumumab in the high-dose model, reflecting its stronger ability to induce EGF-R downmodulation and growth inhibition. In the low-dose model, zalutumumab and panitumumab similarly prevented tumor growth. Thus, our results identify myeloid cell-mediated ADCC as a potent and additional mechanism of action for EGF-R-directed immunotherapy.

Selection of recombinant IgE antibodies binding the β-lactoglobulin allergen in a conformation-dependent manner

Cow's milk allergy (CMA) is a common food allergy, especially among infants and young children. Approximately 85% of milk-allergic children outgrow their allergy by the age of three but the remaining 15% remain allergic. Bovine β-lactoglobulin (BLG) is one of the major allergens in cow's milk. There is a definite need for the specific and sensitive detection of allergenic substances. Validated methods are obligatory to demonstrate allergen contamination and even fatal hidden allergens and, thus, to prevent life-threatening conditions of allergic persons. In this study, we constructed human IgE scFv libraries from an adult milk-allergic patient and isolated the first recombinant IgE antibodies specific to a food allergen, BLG. The selection of the IgE antibody libraries with two distinct panning procedures resulted in the enrichment of four clones having different BLG-binding profiles; two of the clones recognize the native BLG whereas the other two recognize only the heat-denatured form of BLG. For further characterization, the scFv fragments were converted to Fab fragments with human IgG1 isotype. The D1 Fab fragment, binding native BLG with nanomolar affinity, also partially inhibited serum IgE binding to BLG. These BLG-specific IgE antibodies can be applied for the detection of both native and denatured BLG in cow's milk products and furthermore, for the optimization of manufacturing processes to develop safe hypoallergenic milk products.

Regulation of Antibody-Dependent Cellular Cytotoxicity by IgG Intrinsic and Apparent Affinity for Target Antigen

Unconjugated mAbs have emerged as useful cancer therapeutics. Ab-dependent cellular cytotoxicity (ADCC) is believed to be a major antitumor mechanism of some anticancer Abs. However, the factors that regulate the magnitude of ADCC are incompletely understood. In this study, we described the relationship between Ab affinity and ADCC. A series of human IgG1 isotype Abs was created from the anti-HER2/neu (also named c-erbB2) C6.5 single-chain Fv (scFv) and its affinity mutants. The scFv affinities range from 10(-7) to 10(-11) M, and the IgG Abs retain the affinities of the scFv from which they were derived. The apparent affinity of the Abs ranged from nearly 10(-10) M (the lowest affinity variant) to almost 10(-11) M (the other variants). The IgG molecules were tested for their ability to elicit ADCC in vitro against three tumor cell lines with differing levels of HER2/neu expression using unactivated human PBMC from healthy donors as the effector cells. The results demonstrated that both the apparent affinity and intrinsic affinity of the Abs studied regulate ADCC. High-affinity tumor Ag binding by the IgGs led to the most efficient and powerful ADCC. Tumor cells expressing high levels of HER2/neu are more susceptible to the ADCC triggered by Abs than the cells expressing lower amounts of HER2/neu. These findings justify the examination of high affinity Abs for ADCC promotion. Because high affinity may impair in vivo tumor targeting, a careful examination of Ab structure to function relationships is required to develop optimized therapeutic unconjugated Abs.