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Principal Investigator: adrian u wiestner
Organization: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
Fiscal Year: 2022
Award: $1,663,921
Funding agency: National Heart Lung and Blood Institute
Monoclonal antibodies (mAbs) combined with chemotherapy improve survival of lymphoma patients. Fc receptor-dependent effector mechanisms achieve tumor cell killing but also induce antigen loss through trogocytosis. Activation of complement can lead to cell lysis and invariably deposits complement C3 activation fragments on the cell surface. For chronic lymphocytic leukemia (CLL) it is well established that not all of the mAb-targeted B cells are killed during these reactions. Instead, substantial numbers of surviving B cells that have lost CD20 due to trogocytosis and are covalently tagged with the complement breakdown product C3d are readily demonstrable in blood and bone marrow. We sought to test the hypothesis that covalently attached C3d fragments deposited on B cells by anti-CD20 mAbs constitute a neoantigen suitable for targeting antigen loss variants and, when used in combination with a complement fixing mAb, can increase efficacy of mAb therapy. We generated mouse and rabbit mAbs specific for C3d and selected three mAbs that bind distinct epitopes to construct human IgG1 chimeric mAbs. For proof-of-concept, we demonstrated binding of a murine anti-C3d mAb to CLL cells from patients obtained 24 hours after ofatumumab administration; these cells had lost CD20 and carried covalently-bound C3d. The anti-C3d mAb effectively killed these cells in a patient-derived xenograft mouse model. In subsequent in vivo studies, we used rabbit human IgG1 chimeric mAbs that cross-react with murine C3d. In lymphoma xenografts, co-administration of anti-C3d mAbs with either rituximab or ofatumumab was more effective than the anti-CD20 mAb alone: in HBL xenografts median survival increased from 35 days with ofatumumab alone to 63 days for the combination (P=0.012). In SUDHL6 xenografts, median survival was 114 days for anti-CD20 (either rituximab or ofatumumab) treated mice and not reached for mice treated with anti-CD20 combined with an anti-C3d mAb (P=0.008), and tumor-free survival at 6 months was 20% for mice treated with an anti-CD20 mAb alone and 75% for mice treated with the combination. In surviving mice, there was no evidence of any adverse side effects or tissue damage. To extend the application of this invention to Multiple Myeloma (MM) we investigated the combination of the anti-C3d mAbs with the anti-CD38 mAb daratumumab (DARA) that is widely used in patients with MM. Rapid reduction of surface CD38 levels in MM cells following DARA infusion due to trogocytosis has been demonstrated in patients. DARA is a strong activator of complement and all cells reacted with daratumumab are opsonized by C3d. To test the combination of DARA with anti-C3d mAbs in vivo, we used Balb/c-SCID mice bearing subcutaneous MM1-R tumors that were randomly divided into different treatment groups: DARA single agent (n=7), or combination of DARA + anti-C3d antibody (n=26), starting on day 7 post tumor challenge and continued for 6 weeks. Mice were euthanized when tumors reached pre-defined limits. Mice treated with DARA (20mg/kg) alone had median survival of 43 days and two mice survived to day 175. Median survival for mice treated with DARA (10mg/kg) plus an anti-C3d mAb (10mg/kg) was 162 days (P = 0.006), and 13 of 26 (50%) mice in this cohort survived tumor-free beyond 200 days. All the surviving mice showed no evidence of tumor, weight loss or other morbidity indicating that they are essentially tumor-free. In summary, these results demonstrate that combination treatment of DARA with an anti-C3d mAb improves efficacy over single agent DARA leading to long-term survival in the MM.1R xenograft model. The combination of complement targeting mAbs with DARA could improve clinical efficacy and overcome resistance to single agent therapy. In conclusion, our findings indicate that C3d-targeting with a specific mAb can provide a decisive second hit to enhance the efficacy of complement fixing mAbs commonly used in lymphoma and MM therapy.
We developed a novel CD19/CD3 bsAb in the single-chain Fv-Fc format (CD19/CD3-scFv-Fc) with a half-life of approximately 5 days. In the NOD/SCID/IL2Rnull patient-derived xenograft mouse model, once-weekly treatment with CD19/CD3-scFv-Fc eliminated >98% of treatment-nave CLL cells in blood and spleen. CD19/CD3-scFv-Fc induced more rapid killing of CLL cells from ibrutinib-treated patients than those from treatment-nave patients. CD19/CD3-scFv-Fc also demonstrated potent activity against CLL cells from patients with acquired ibrutinib-resistance harboring BTK and/or PLCG2 mutations in vitro and in vivo using patient-derived xenograft models. Compared to observations with samples from treatment-nave patients, T cells from patients being treated with ibrutinib expanded more rapidly and exerted superior cytotoxic activity in response to the bsAb. BTKi therapy transcriptionally downregulated immunosuppressive effectors expressed by CLL cells, including CTLA-4 and CD200. CTLA-4 blockade with ipilimumab in vitro increased the cytotoxic activity of the bsAb in BTKi-nave but not BTKi-treated PBMCS. Taken together, BTKis enhance bsAb induced cytotoxicity by relieving T cells of immunosuppressive restraints imposed by CLL cells. The benefit of combining bsAb immunotherapy with BTKis needs to be confirmed in clinical trials. We are currently expanding our pre-clinical studies to testing the CD20/CD3 bsAb epcoritamab.
CLL patients have reduced antibody responses to vaccines. In a clinical trial, we measured the serologic and cellular immunogenicity of the recombinant zoster vaccine (RZV) in CLL patients who were treatment nave (TN) or receiving Bruton tyrosine kinase inhibitor (BTKi) therapy. The primary endpoint was antibody response to RZV (4-fold increase in anti-glycoprotein E (gE) antibody titer). Cellular response of gE-specific CD4+ T cells was assessed by flow cytometry for intracellular staining for upregulation of 2 effector molecules. The antibody response rate was significantly higher in the TN cohort (76.8% compared to patients receiving a BTKi (40.0%; P = .0002). The cellular response rate was also significantly higher in the TN cohort (70.0% compared to the BTKi group (41.3%; P = .0072). A concordant positive serologic and cellular immune response was observed in 69.1% of subjects. Serologic titers and T cell responses were not correlated with age, absolute B and T cell counts, or serum immunoglobulin levels (all P > 0.05). As treatment with BTKi can suppress vaccine responses, we investigated whether BTKi interruption around the time of a Covid-19 booster injection could be beneficial. We studied 86 patients enrolled on clinical trials at the NIH who received Covid-19 mRNA vaccines. After the primary series, seroconversion (anti-spike 0.8 U/mL) was detected in 53% of BTKi-treated patients, 57% of patients on single-agent venetoclax (VEN), and 67% of treatment-naive (TN) patients. After booster, anti-spike antibody was detected in 87% of BTKi-treated patients, 50% of patients on single-agent VEN, and 83% of TN patients. Of the patients who did not respond to the primary series, 57% seroconverted post booster. Of patients on BTKi, 21/40 (52%) had seroconversion after primary series, albeit with mostly weak responses. After booster, 34/40 (85%) had detectable anti-spike Ab. Twelve patients interrupted BTKi for a median of 21 days (range 8-22) around the time of booster. Patients who interrupted BTKi had higher anti-spike Ab (median 7,148 U/mL, IQR) than those who continued therapy (median 1,198 U/mL, IQR, p=0.018). Three patients experienced lymph node pain and swelling during BTKi interruption and resumed BTKi earlier than intended. In conclusion, BTKi interruption at the time of vaccination results in a more robust Ab response.
Terms: <Ab response><Agammaglobulinaemia tyrosine kinase><Age><Antibodies><Antibody Formation><Antibody Production><Antibody Response><Antibody Therapy><Antibody titer measurement><Antigen Targeting><Antigenic Determinants><Antigens><B blood cells><B cell><B cell progenitor kinase><B cells><B-Cell CLL><B-Cell Chronic Lymphocytic Leukemia><B-Cell Chronic Lymphogenous Leukemia><B-Cell Chronic Lymphoid Leukemia><B-Cell Lymphocytic Leukemia><B-Cells><B-Lymphocytes><B-Lymphocytic Leukemia><B-cell><BALB C Mouse><BALB/c><Bi-specific antibodies><Bifunctional Antibodies><Binding><Binding Determinants><Bispecific Antibodies><Blood><Blood Reticuloendothelial System><Blood Serum><Body Tissues><Body Weight decreased><Bone Marrow><Bone Marrow Reticuloendothelial System><Bp35><Bruton's tyrosine kinase><C2B8 Monoclonal Antibody><C3 d><C3d><CD 200><CD152><CD152 Antigen><CD152 Gene><CD19><CD19 gene><CD20><CD200><CD3><CD3 Antigens><CD3 Complex><CD3 molecule><CD4 Cells><CD4 Positive T Lymphocytes><CD4 T cells><CD4 helper T cell><CD4 lymphocyte><CD4+ T-Lymphocyte><CD4-Positive Lymphocytes><COVID-19><COVID-19 booster><COVID-19 vaccine booster><COVID19><CTLA 4><CTLA-4 Gene><CTLA-4 blockade><CTLA4><CTLA4 blockade><CTLA4 gene><CTLA4-TM><CV-19><CV19><Cell Body><Cell Count><Cell Number><Cell surface><Cells><Chronic B-Lymphocytic Leukemia><Chronic Lymphatic Leukemia><Chronic Lymphoblastic Leukemia><Chronic Lymphocytic Leukemia><Chronic Lymphogenous Leukemia><Clinical Treatment Moab><Clinical Trials><Complement><Complement 3d><Complement Activation><Complement C3d><Complement Proteins><Conduct Clinical Trials><Cytolysis><Cytotoxic T-Lymphocyte Protein 4><Cytotoxic T-Lymphocyte-Associated Antigen 4><Cytotoxic T-Lymphocyte-Associated Protein 4><Cytotoxic T-Lymphocyte-Associated Serine Esterase-4><Deposit><Deposition><Domestic Rabbit><Enrollment><Epitopes><Fc Receptor><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Gene Transcription><Genetic Alteration><Genetic Change><Genetic Transcription><Genetic defect><Germinoblastic Sarcoma><Germinoblastoma><Glycoproteins><Half-Life><Heterograft><Heterologous Transplantation><Hour><Human><IgG1><Immune Globulins><Immune mediated therapy><Immune response><Immunoglobulins><Immunological response><Immunologically Directed Therapy><Immunotherapy><In Vitro><Inbred BALB C Mice><Infusion><Infusion procedures><Injections><Innovative Therapy><Interruption><Lead><Leu-16><Lymph Node Reticuloendothelial System><Lymph node proper><Lymphatic nodes><Lymphoma><Lysis><Lytotoxicity><MS4A1><MS4A1 gene><MS4A2><MabThera><Malignant Lymphoma><Malignant lymphoid neoplasm><Measures><Mice><Mice Mammals><MoAb CD20><Modern Man><Molecular Interaction><Monoclonal Antibodies><Monoclonal Antibody CD20><Monoclonal Antibody Therapy><Morbidity><Morbidity - disease rate><Multiple Myeloma><Murine><Mus><Mutation><NIH><National Institutes of Health><OKT3 antigen><Oryctolagus cuniculus><PBMC><PDX model><PLCG2><PLCG2 gene><PTK Inhibitors><Pain><Painful><Patient derived xenograft><Patients><Pb element><Peripheral Blood Mononuclear Cell><Phosphatidylinositol-Specific Phospholipase C><Phospholipase C Gamma 2><Plasma-Cell Myeloma><Protein Tyrosine Kinase Inhibitors><RNA Expression><RNA vaccine><RNA-based vaccine><Rabbits><Rabbits Mammals><Reaction><Recombinants><Resistance><Reticulolymphosarcoma><Rituxan><Role><SARS-CoV-2 booster><SARS-CoV-2 vaccine booster><SCID Mice><Sampling><Series><Serology><Serum><Severe Combined Immunodeficient Mice><Spleen><Spleen Reticuloendothelial System><Staining method><Stains><Surface><Swelling><T cell response><T-Cells><T-Lymphocyte><T3 Antigens><T3 Complex><T3 molecule><T4 Cells><T4 Lymphocytes><TK Inhibitors><Testing><Time><Tissues><Transcription><Tumor Cell><Tumor Weights><Tyrosine Kinase Inhibitor><United States National Institutes of Health><Up-Regulation><Upregulation><Vaccines><Variant><Variation><Weight Loss><Weight Reduction><Xenograft><Xenograft Model><Xenograft procedure><Xenotransplantation><Yervoy><Zoster Vaccine><ages><anti-CD20><antibody based therapies><antibody biosynthesis><antibody receptor><antibody titering><antibody treatment><antibody-based therapeutics><antibody-based treatment><biological adaptation to stress><body weight loss><booster against COVID-19><booster against SARS-CoV-2><bpk protein><bsAb><btk protein><cell killing><chemotherapy><chronic lymphocytic leukemia cell><chronic lymphoid leukemia><clinical efficacy><cohort><complement pathway regulation><corona virus disease 2019><coronavirus disease 2019><coronavirus disease-19><coronavirus infectious disease-19><cytotoxic><cytotoxic T-lymphocyte antigen 4><cytotoxicity><enroll><experience><flow cytophotometry><genome mutation><heavy metal Pb><heavy metal lead><host response><immune system response><immune therapeutic approach><immune therapeutic interventions><immune therapeutic regimens><immune therapeutic strategy><immune therapy><immune-based therapies><immune-based treatments><immuno therapy><immunogen><immunogenicity><immunoglobulin biosynthesis><immunoresponse><improved><in vivo><inhibitor drug><inhibitor therapeutic><inhibitor therapy><invention><ipilimumab><lymph gland><lymph nodes><lymphnodes><lymphoid cancers><lymphoid malignancy><mAb therapy><mAbs><mRNA vaccine><mRNA-based vaccine><mouse model><murine model><myeloma><myelomatosis><neo-antigen><neo-epitopes><neoantigens><neoepitopes><neoplastic cell><new approaches><novel><novel approaches><novel strategies><novel strategy><outcome following vaccination><outcome following vaccine><patient derived xenograft model><pre-clinical study><preclinical study><primary end point><primary endpoint><reaction; crisis><resistant><response><restraint><result following vaccination><result following vaccine><rituximab><seroconversion><side effect><social role><stress response><stress; reaction><subcutaneous><subdermal><thymus derived lymphocyte><treatment group><tumor><vaccination outcome><vaccination result><vaccine outcome><vaccine response><vaccine responsiveness><vaccine result><vaccine-induced response><wt-loss><xeno-transplant><xeno-transplantation><xenograft transplant model><xenotransplant model>