If immunized with an antigen of interest, transgenic mice with large portions of unrearranged human immunoglobulin loci can produce fully human antigen-specific antibodies; several such antibodies ...are in clinical use. However, technical limitations inherent to conventional transgenic technology and sequence divergence between the human and mouse immunoglobulin constant regions limit the utility of these mice. Here, using repetitive cycles of genome engineering in embryonic stem cells, we have inserted the entire human immunoglobulin variable-gene repertoire (2.7 Mb) into the mouse genome, leaving the mouse constant regions intact. These transgenic mice are viable and fertile, with an immune system resembling that of wild-type mice. Antigen immunization results in production of high-affinity antibodies with long human-like complementarity-determining region 3 (CDR3H), broad epitope coverage and strong signatures of somatic hypermutation. These mice provide a robust system for the discovery of therapeutic human monoclonal antibodies; as a surrogate readout of the human antibody response, they may also aid vaccine design efforts.
Age-related macular degeneration (AMD) is a leading cause of legal blindness in the Western world. There are effective treatments for the vascular complications of neo-vascular AMD, but no effective ...therapies are available for the dry/atrophic form of the disease. A previously described transgenic CFH-gene deficient mouse model, (cfh-/-), shows hallmarks of early AMD. The ocular phenotype has been further analysed to demonstrate amyloid beta (Aβ) rich basement membrane deposits associated with activated complement C3. Cfh-/- mice were treated systemically in both prophylactic and therapeutic regimes with an anti-Aβ monoclonal antibody (mAb), 6F6, to determine the effect on the cfh-/- retinal phenotype. Prophylactic treatment with 6F6 demonstrated a dose dependent reduction in the accumulation of both Aβ and activated C3 deposition. A similar reduction in the retinal endpoints could be seen after therapeutic treatment. Serum Aβ levels after systemic administration of 6F6 show accumulation of Aβ in the periphery suggestive of a peripheral sink mechanism. In summary, anti-Aβ mAb treatment can partially prevent or reverse ocular phenotypes of the cfh-/- mouse. The data support this therapeutic approach in humans potentially modulating two key elements in the pathogenesis of AMD - Aβ and activated, complement C3.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recombinant erythropoietin (EPO) and iron substitution are a standard of care for treatment of anemias associated with chronic inflammation, including anemia of chronic kidney disease. A black box ...warning for EPO therapy and concerns about negative side effects related to high-dose iron supplementation as well as the significant proportion of patients becoming EPO resistant over time explains the medical need to define novel strategies to ameliorate anemia of chronic disease (ACD). As hepcidin is central to the iron-restrictive phenotype in ACD, therapeutic approaches targeting hepcidin were recently developed. We herein report the therapeutic effects of a fully human anti-BMP6 antibody (KY1070) either as monotherapy or in combination with Darbepoetin alfa on iron metabolism and anemia resolution in 2 different, well-established, and clinically relevant rodent models of ACD. In addition to counteracting hepcidin-driven iron limitation for erythropoiesis, we found that the combination of KY1070 and recombinant human EPO improved the erythroid response compared with either monotherapy in a qualitative and quantitative manner. Consequently, the combination of KY1070 and Darbepoetin alfa resulted in an EPO-sparing effect. Moreover, we found that suppression of hepcidin via KY1070 modulates ferroportin expression on erythroid precursor cells, thereby lowering potentially toxic-free intracellular iron levels and by accelerating erythroid output as reflected by increased maturation of erythrocyte progenitors. In summary, we conclude that treatment of ACD, as a highly complex disease, becomes more effective by a multifactorial therapeutic approach upon mobilization of endogenous iron deposits and stimulation of erythropoiesis.
•Combined anti-BMP6 antibody and EPO treatment improves systemic iron availability and reduces EPO needs for the treatment of ACD.•Modulation of ferroportin on erythroid precursors results in reduced free intracellular iron levels and maturation of the erythroid lineage.
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As the number of antibody drugs being approved and marketed increases, our knowledge of what makes potential drug candidates a successful product has increased tremendously. One of the critical ...parameters that have become clear in the field is the importance of mAb “developability.” Efforts are being increasingly focused on simultaneously selecting molecules that exhibit both desirable biological potencies and manufacturability attributes. In the current study mutations to improve the developability profile of a problematic antibody that inconsistently precipitates in a batch scale‐dependent fashion using a standard platform purification process are described. Initial bioinformatic analysis showed the molecule has no obvious sequence or structural liabilities that might lead it to precipitate. Subsequent analysis of the molecule revealed the presence of two unusual positively charged mutations on the light chain at the interface of VH and VL domains, which were hypothesized to be the primary contributor to molecule precipitation during process development. To investigate this hypothesis, straightforward reversion to the germline of these residues was carried out. The resulting mutants have improved expression titers and recovered stability within a forced precipitation assay, without any change to biological activity. Given the time pressures of drug development in industry, process optimization of the lead molecule was carried out in parallel to the “retrospective” mutagenesis approach. Bespoke process optimization for large‐scale manufacturing was successful. However, we propose that such context‐dependent sequence liabilities should be included in the arsenal of in silico developability screening early in development; particularly since this specific issue can be efficiently mitigated without the requirement for extensive screening of lead molecule variants.
In this article context‐dependent sequence liabilities were mapped using structural modelling to improve the developability of a problematic mAb. As few as 3 point mutants were made and tested using this methodology to allow rapid and pragmatic lead optimization.
Background:
Hemophilia A is caused by deficiency of factor VIII (F.VIII), an essential blood-clotting cofactor. The current standard of care involves prophylactic or on-demand use of F.VIII. However, ...approximately 30% of severe hemophilia A patients develop inhibitory antibodies against the replacement factor VIII. Additionally, venous infusion of F.VIII is burdensome especially in pediatric patients. Hemlibra®, a humanized bispecific antibody (BiAb) that mimics the action of factor VIII, has been developed and approved for treating hemophilia A patients with or without inhibitors. Using Kymab's IntelliSelect® Bispecific antibody discovery platform, a fully human common light chain (CLC) bispecific antibody was developed and optimized to mimic the function of F.VIII. The biological activity of the CLC bispecific antibody was characterized using clinically relevant hemostatic assays.
Methods:
Kymab IntelliSelect® Transgenic mice were immunized with human factor IX (F.IX) or factor X (F.X). Isolated F.IX and F.X arms were combinatorially expressed as 2-heavy-2-light (2H2L) BiAbs. Light chain of a biologically active F.IX arm was chosen to generate CLC transgenic mice. CLC transgenic mice were immunized with F.X to identify F.X heavy chains pairing with CLC. The isolated F.X heavy chains were co-expressed with the heavy and light chains of the selected F.IX arm as CLC BiAbs and re-screened by functional assays. Biologically active BiAbs were further optimized to generate a lead BiAb, KY1049. KY1049 heterodimer was purified by a two-step method using Protein A and cation ion exchange chromatography (cIEX). The purified BiAb was analyzed by analytical HPLC and mass spectrometry (MS). The purified BiAb was also characterized using a combination of in vitro and ex vivo hemostatic assays including chromogenic FXase (FXase), activated partial thromboplastin time (aPTT) and thrombin generation assay (TGA). Binding of KY1049 to F.IX and F.X was studied using surface plasmon resonance (SPR).
Results:
More than 8,000 2H2L BiAbs were first screened by high-throughput chromogenic FXase assay. Functionally active 2H2L BiAbs were identified, consisting of one F.IX arm and different F.X arms. The light chain of this promiscuous F.IX arm was chosen to generate CLC transgenic mice expressing the light chain exclusively. More than 400 F.X heavy chains isolated from the CLC transgenic mice were screened to identify functionally active CLC BiAbs. Further work to iteratively and combinatorially optimize the variable domains resulted in KY1049, a lead BiAb which demonstrates robust hemostatic activity. KY1049 demonstrates a dose-dependent reduction in clotting time and a dose-dependent increase in thrombin burst, thereby functionally restoring the hemostatic activity of F.VIII-depleted plasma. Compared with a sequence identical analog (SIA) of Hemlibra®, KY1049 shows comparable efficacy in a range of concentration of IgG (0.1 to 300 nM). Importantly, KY1049 can be purified using a routine purification process. The purity and the identity of KY1049 was confirmed by mass spectrometry. KY1049 was shown to simultaneously bind F.IX and F.X using SPR in confirmation of its mode of action.
Summary/conclusions:
KY1049, developed using Kymab's IntelliSelect® Bispecific platform, is a potent F.VIII mimetic bispecific antibody with activities comparable to a SIA of Hemlibra®. This fully human CLC bispecific antibody can potentially provide a new treatment option for hemophilia A patients.
Wang:Kymab Ltd.: Employment, Equity Ownership, Patents & Royalties. Blackwood:Kymab Ltd.: Employment, Equity Ownership, Patents & Royalties. Magliozzi:Kymab Ltd.: Employment, Equity Ownership, Patents & Royalties. Moraes:Kymab Ltd.: Employment, Equity Ownership. Hollins:Kymab Ltd.: Employment, Equity Ownership. Sinopoli:Kymab Ltd.: Employment, Equity Ownership. Chi:Kymab Ltd.: Employment, Equity Ownership. Mitchell:Kymab Ltd.: Employment, Equity Ownership. Sellick:Kymab Ltd.: Employment, Equity Ownership. Pearce:Kymab Ltd.: Employment, Equity Ownership. Theurl:Kymab Ltd.: Consultancy, Equity Ownership; Sierra Oncoloy: Research Funding. Germaschewski:Kymab Ltd.: Employment, Equity Ownership. Galson:Kymab Ltd.: Employment, Equity Ownership. Badiali:Kymab Ltd.: Employment, Equity Ownership. Dickson:Kymab Ltd.: Employment, Equity Ownership. Bradley:Kymab Ltd.: Employment, Equity Ownership, Patents & Royalties. Lee:Kymab Ltd.: Employment, Equity Ownership, Patents & Royalties.
Beta thalassemia is an inherited hemoglobinopathy caused by a genetic defect in beta-globin gene and characterised by ineffective erythropoiesis, iron overload, splenomegaly and anemia. Excessive EPO ...production, resulting from the anemia, has a suppressive effect on the iron regulator hepcidin leading to increased iron absorption from the gut and release from internal stores. This in turn leads to tissue iron overload in transfusion independent (NTDT) patients and exacerbates the situation in transfusion dependent (TDT) patients. Primary standard of care treatment for TDT patients involves regular blood transfusions resulting in secondary iron overload and toxic tissue damage caused by non-transferrin bound iron (NTBI), which requires iron chelator treatment. Although transfusions and iron chelation have improved TDT patient prognosis, the iron overload experienced by some patients today still represents an unmet clinical need. The role of TMPRSS6 in iron regulation is already well established and reducing Tmprss6 expression was shown to increase hepcidin expression, correcting the iron overload, splenomegaly and anemia in Hbbth3/+ mice (Guo et al, JCI, 2013).
Here we describe a first-in-class antibody targeting MTP-2, termed KY1066, for the treatment of diseases of iron overload, such as beta thalassemia. Through immunising mice transgenic for human immunoglobin heavy and light chain variable regions with purified human MTP-2 extra-cellular domain (ECD) and full-length human MTP-2 expressed on cells, we were able to obtain cross-reactive, MTP-2-specific monoclonal antibodies (mAbs). Our lead molecule, KY1066, was found to be a cross-reactive neutraliser of MTP-2 enzymatic activity both in vitro and in vivo and mechanistically blocks MTP-2 through binding to the serine protease (SP) domain active site, preventing cleavage of substrates. As a result, it was seen to increase BMP/SMAD signalling and elevate levels of hepcidin expression from hepatocyte cells following a single I.P. dose (10 mg/kg) in C57BL/6J mice. The elevation of hepcidin decreased serum iron and transferrin saturation through increased internalisation and degradation of ferroportin. In Hbbth3/+ mice, a single I.P. dose (10 mg/kg) showed a decrease of 52% and 47% in serum iron and transferrin saturation (TSAT) respectively, at the 2-week timepoint. Furthermore, with repeat dosing, we were able to show consistent iron restriction in a Hbbth3/+ mice over multiple weeks. Together this provides evidence KY1066 has the potential to treat iron overloaded patients, such as in beta thalassemia, to improve the anemia and reduce the transfusion and iron chelation need.
Wake:Kymab Ltd: Employment, Equity Ownership. Papworth:Kymab Ltd: Employment, Equity Ownership. Bayliss:Kymab Ltd: Employment, Equity Ownership. Grimshaw:Kymab Ltd: Employment, Equity Ownership. Rynkiewicz:Kymab Ltd: Employment, Equity Ownership. Paterson:Kymab Ltd: Employment, Equity Ownership. Poindron:Kymab Ltd: Employment, Equity Ownership. Carter:Kymab Ltd: Employment. Hudson:Kymab Ltd: Employment, Equity Ownership. Theurl:Kymab Ltd: Consultancy, Equity Ownership. Germaschewski:Kymab Ltd.: Employment, Equity Ownership. Meynard:Kymab Ltd: Research Funding.
Anemia of chronic disease (ACD) is the most common cause of anemia in hospitalized patients. The underlying pathophysiological mechanisms are manifold, including reduced Erythropoietin (EPO) ...availability and sensitivity, direct negative effects of inflammatory cytokines on erythropoiesis and functional iron deficiency due to iron restriction mainly in the reticuloendothelial system (RES). The latter can be ascribed to increased hepcidin levels, a small liver derived peptide that has been found to be the key iron regulator.
Up to date, most patients suffering from ACD are treated with a combination of both ESA (Erythropoietin Stimulating Agent) and intravenous (i.v.) iron to maintain hemoglobin (Hb) levels. Despite this combination therapy a significant number of patients require increasing doses of i.v. iron and ESA during their medical history - often resulting in continuous, potential toxic iron overload and ESA doses that exceed the acceptable range. As hepcidin is central to the iron-restrictive phenotype in ACD, several therapeutic approaches of hepcidin modulation have been investigated to overcome iron overload and EPO resistance. Some of these therapies are currently investigated in early clinical trials.
We here report the effects of a fully human anti-BMP6 antibody on anemia, iron metabolism, erythropoiesis and ESA dosing in two different, well established rodent models of ACD.
As BMPs, mainly BMP2 and BMP6, have been reported to be involved in hepcidin control, with knock out mice showing very low hepcidin levels even in an inflammatory setting, a fully human anti-BMP6 antibody has been developed to suppress hepcidin levels.
We tested the antibody treatment in two distinct ACD animal models: First, a rat arthritis model (PG-APS in Lewis rats) and second, a Chronic Kidney Disease (CKD) mouse anemia model (Adenine model in C57BL/6 mice). Both models present with long-lasting anemia as seen in humans suffering from ACD. Mice and rats were treated with different doses of the anti-BMP6 antibody with and without ESA. Whole blood count, serum iron parameters (including hepcidin), bone marrow erythropoiesis determined by FACS analysis, cytokine expression and iron metabolism gene expression in spleen, liver and kidney were analyzed.
Anti-BMP6 as well as ESA monotherapy resulted in a net increase in Hb level but only anti-BMP6 treatment significantly increased MCV and MCH, which can be ascribed to effective iron mobilization. In contrast, ESA therapy raised Hb levels by increasing red blood cell numbers. Of note, mere i.v. iron supplementation (sodium ferric gluconate), even at high doses, was not able to restore Hb levels to the same extent as the anti-BMP6 monotherapy.
Strikingly, combination of both, ESA and anti-BMP6 treatment, had a synergistic effect on Hb levels, especially in the rat PG-APS model. Combination therapy of low ESA doses that only had a modest effect as a monotherapy, led to a dramatic increase in Hb levels, even exceeding those seen in healthy rats. Based on these results, additional experiments were performed to investigate the potential of this combination treatment to reduce ESA doses. Indeed, when anti-BMP6 was combined with a significantly reduced total ESA dose Hb levels were corrected to normal values in disease animals. Anti-BMP6 treatment also led to a significant decrease of iron deposition in the spleen with no iron deposition in parenchymal organs, indicating that the freed-up iron was effectively used for erythropoiesis and not just distributed elsewhere.
In summary, anti-BMP6 therapy worked synergistically with ESA treatment in two different models of ACD leading to significantly increased Hb levels, a reduced ESA need as well as reduced iron overload in the RES. Furthermore, these experiments clearly show that treatment of ACD, being a complex multifactorial disease, benefits from using a combination of diversified approaches to overcome anemia and significantly reduce the dose of each therapeutic.
Wake:Kymab Ltd.: Employment. Bayliss:Kymab Ltd.: Employment. Papworth:Kymab Ltd.: Employment. Carvalho:Kymab Ltd.: Employment. Deantonio:Kymab Ltd.: Employment. Weiss:Kymab Ltd.: Consultancy. Germaschewski:Kymab Ltd.: Employment. Theurl:Kymab Ltd.: Consultancy, Research Funding.
Pulmonary arterial hypertension (PAH) is a rare but fatal disease. Current treatments increase life expectancy but have limited impact on the progressive pulmonary vascular remodelling that drives ...PAH. Osteoprotegerin (OPG) is increased within serum and lesions of patients with idiopathic PAH and is a mitogen and migratory stimulus for pulmonary artery smooth muscle cells (PASMCs). Here, we report that the pro-proliferative and migratory phenotype in PASMCs stimulated with OPG is mediated via the Fas receptor and that treatment with a human antibody targeting OPG can attenuate pulmonary vascular remodelling associated with PAH in multiple rodent models of early and late treatment. We also demonstrate that the therapeutic efficacy of the anti-OPG antibody approach in the presence of standard of care vasodilator therapy is mediated by a reduction in pulmonary vascular remodelling. Targeting OPG with a therapeutic antibody is a potential treatment strategy in PAH.
Abstract
The last few years have shown a strong paradigm shift in cancer therapies with the approval of antibodies targeting immune checkpoints (CTLA-4, PD-1 and PD-L1). These immune checkpoint ...blockers (ICBs) are associated with a strong and long-lasting response in patients suffering from different malignancies. However, there is still a high proportion of patients showing intrinsic or acquired resistance to ICBs. Although the mechanisms associated with the lack of immune response are multiple, the presence of highly anti-inflammatory regulatory T cells (TRegs) in the tumour microenvironment (TME) is known to negatively affect the response to these therapies. Similarly, high TReg levels in the TME have been associated with poor prognosis in several cancers. Together, this highlights the potential of targeting and depleting TRegs to enhance anti-tumour responses.
The Inducible T-cell costimulator (ICOS/CD278) is related to the CD28 superfamily and is induced when T cells get activated. ICOS expression levels vary in different immune cell subtypes and in different tissues. In preclinical mouse tumour models, TRegs (CD4+/FOXP3+) constitutively express ICOS on their surface and the expression of ICOS on TRegs is significantly higher than that on effector T cells (TEffs). In addition, ICOS expression on TRegs is higher in the TME than in the blood or spleen, which makes it a strong candidate for preferential depletion of TRegs in tumours. By immunizing Kymice™ in which the endogenous Icos gene has been knocked out, we identified a novel, fully human antibody called KY1044 that cross-reacts with mouse ICOS. KY1044 is an anti-ICOS subclass G1 kappa monoclonal antibody that selectively binds to dimeric ICOS (Fc fusion) with an affinity of less than 2nM. Using in vitro and in vivo approaches we demonstrate that KY1044 has a dual mechanism of action: (1) it promotes the preferential depletion of intratumoural ICOShigh TRegs resulting in an increase in the TEff:TReg ratio in the TME; and (2) it stimulates ICOSLow TEff cells. Using the mouse effector enabled version of KY1044 (mIgG2a) we confirm, using syngeneic models, a strong anti-tumour efficacy as monotherapy or in combination with surrogates of ICBs. We also demonstrated a tumour antigen specific immunity, as highlighted by the rejection of the original tumour cells in animals cured of the disease and re-challenged by the same cell line. Noteworthy, Pharmacodynamic studies demonstrate long-term depletion of TRegs and a significant increase in the TEff:TReg ratio in response to KY1044. In summary, our data demonstrate that targeting ICOS with KY1044 is a valid approach for manipulating the immune system and for inducing a strong anti-tumour response in several indications. The data presented here also warrant the assessment of KY1044 in cancer patients in a clinical trial.
Citation Format: Richard C. Sainson, Matthew McCourt, Anil Thotakura, Nahida Parveen, Miha Kismac, Gwenoline Borhis, Joana Carvalho, Tracey Myers, Robert Rowlands, Hanif Ali, Hannah Craig, Vivian Wong, Qi Liang, Volker Germaschewski. The combination of immune checkpoint blockers with the anti-ICOS KY1044 antibody results in a strong tumor response abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2792.
The immunosuppressive tumor microenvironment constitutes a significant hurdle to immune checkpoint inhibitor responses. Both soluble factors and specialized immune cells, such as regulatory T cells ...(Treg), are key components of active intratumoral immunosuppression. Inducible costimulatory receptor (ICOS) can be highly expressed in the tumor microenvironment, especially on immunosuppressive Treg, suggesting that it represents a relevant target for preferential depletion of these cells. Here, we performed immune profiling of samples from tumor-bearing mice and patients with cancer to demonstrate differential expression of ICOS in immune T-cell subsets in different tissues. ICOS expression was higher on intratumoral Treg than on effector CD8 T cells. In addition, by immunizing an
knockout transgenic mouse line expressing antibodies with human variable domains, we selected a fully human IgG1 antibody called KY1044 that bound ICOS from different species. We showed that KY1044 induced sustained depletion of ICOS
T cells but was also associated with increased secretion of proinflammatory cytokines from ICOS
effector T cells (T
). In syngeneic mouse tumor models, KY1044 depleted ICOS
Treg and increased the intratumoral T
:Treg ratio, resulting in increased secretion of IFNγ and TNFα by T
cells. KY1044 demonstrated monotherapy antitumor efficacy and improved anti-PD-L1 efficacy. In summary, we demonstrated that using KY1044, one can exploit the differential expression of ICOS on T-cell subtypes to improve the intratumoral immune contexture and restore an antitumor immune response.
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