ABSTRACT
Bruton tyrosine kinase inhibitors (BTKis) are a preferred treatment of patients with chronic lymphocytic leukemia (CLL). Indefinite therapy with BTKis, although effective, presents clinical challenges. Combination therapy can deepen responses, shorten treatment duration, and possibly prevent or overcome drug resistance. We previously reported on a CD19/CD3-bispecific antibody (bsAb) that recruits autologous T-cell cytotoxicity against CLL cells in vitro. Compared with observations with samples from treatment-naïve patients, T cells from patients being treated with ibrutinib expanded more rapidly and exerted superior cytotoxic activity in response to the bsAb. In addition to BTK, ibrutinib also inhibits interleukin-2 inducible T-cell kinase (ITK). In contrast, acalabrutinib, does not inhibit ITK. Whether ITK inhibition contributes to the observed immune effects is unknown. To better understand how BTKis modulate T-cell function and cytotoxic activity, we cultured peripheral blood mononuclear cells (PBMCs) from BTKi-naive and ibrutinib- or acalabrutinib-treated CLL patients with CD19/CD3 bsAb in vitro. T-cell expansion, activation, differentiation, and cytotoxicity were increased in PBMCs from patients on treatment with either BTKi compared with that observed for BKTi-naïve patients. BTKi therapy transcriptionally downregulated immunosuppressive effectors expressed by CLL cells, including cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and CD200. CTLA-4 blockade with ipilimumab in vitro increased the cytotoxic activity of the bsAb in BTKi-naïve 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.
Subject(s)
Adenine/analogs & derivatives , Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Antibodies, Bispecific/therapeutic use , Antineoplastic Agents, Immunological/therapeutic use , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Piperidines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Adenine/therapeutic use , Adult , Aged , Aged, 80 and over , Antigens, CD19/immunology , Benzamides/therapeutic use , CD3 Complex/immunology , Female , Humans , Immune Checkpoint Inhibitors/therapeutic use , Ipilimumab/therapeutic use , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Male , Middle Aged , Pyrazines/therapeutic use , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
Antibodies are widely used as cancer therapeutics, but their current use is limited by the low number of antigens restricted to cancer cells. A receptor tyrosine kinase, receptor tyrosine kinase-like orphan receptor 2 (ROR2), is normally expressed only during embryogenesis and is tightly down-regulated in postnatal healthy tissues. However, it is up-regulated in a diverse set of hematologic and solid malignancies, thus ROR2 represents a candidate antigen for antibody-based cancer therapy. Here we describe the affinity maturation and humanization of a rabbit mAb that binds human and mouse ROR2 but not human ROR1 or other human cell-surface antigens. Co-crystallization of the parental rabbit mAb in complex with the human ROR2 kringle domain (hROR2-Kr) guided affinity maturation by heavy-chain complementarity-determining region 3 (HCDR3)-focused mutagenesis and selection. The affinity-matured rabbit mAb was then humanized by complementarity-determining region (CDR) grafting and framework fine tuning and again co-crystallized with hROR2-Kr. We show that the affinity-matured and humanized mAb retains strong affinity and specificity to ROR2 and, following conversion to a T cell-engaging bispecific antibody, has potent cytotoxicity toward ROR2-expressing cells. We anticipate that this humanized affinity-matured mAb will find application for antibody-based cancer therapy of ROR2-expressing neoplasms.
Subject(s)
Antibodies, Monoclonal, Humanized/chemistry , Antibodies, Monoclonal, Humanized/immunology , Antibody Affinity , Receptor Tyrosine Kinase-like Orphan Receptors/immunology , Animals , Antibodies, Monoclonal, Humanized/therapeutic use , Antibody Specificity , CD3 Complex/immunology , Cell Line, Tumor , Crystallization , Humans , Hydrogen-Ion Concentration , Models, Molecular , Protein Domains , RabbitsABSTRACT
T cell-engaging bispecific antibodies (biAbs) present a promising strategy for cancer immunotherapy, and numerous bispecific formats have been developed for retargeting cytolytic T cells toward tumor cells. To explore the therapeutic utility of T cell-engaging biAbs targeting the receptor tyrosine kinase ROR1, which is expressed by tumor cells of various hematologic and solid malignancies, we used a bispecific ROR1 × CD3 scFv-Fc format based on a heterodimeric and aglycosylated Fc domain designed for extended circulatory t1/2 and diminished systemic T cell activation. A diverse panel of ROR1-targeting scFv derived from immune and naïve rabbit antibody repertoires was compared in this bispecific format for target-dependent T cell recruitment and activation. An ROR1-targeting scFv with a membrane-proximal epitope, R11, revealed potent and selective antitumor activity in vitro, in vivo, and ex vivo and emerged as a prime candidate for further preclinical and clinical studies. To elucidate the precise location and engagement of this membrane-proximal epitope, which is conserved between human and mouse ROR1, the 3D structure of scFv R11 in complex with the kringle domain of ROR1 was determined by X-ray crystallography at 1.6-Å resolution.
Subject(s)
Antibodies, Bispecific/immunology , Antineoplastic Agents/immunology , Epitopes/immunology , Receptor Tyrosine Kinase-like Orphan Receptors/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , CD3 Complex/immunology , Cell Line, Tumor , Crystallography, X-Ray/methods , Humans , Immunotherapy/methods , Jurkat Cells , K562 Cells , Mice , Rabbits , Single-Chain Antibodies/immunology , Xenograft Model Antitumor Assays/methodsABSTRACT
The Bruton tyrosine kinase inhibitor ibrutinib induces high rates of clinical response in chronic lymphocytic leukemia (CLL). However, there remains a need for adjunct treatments to deepen response and to overcome drug resistance. Blinatumomab, a CD19/CD3 bispecific antibody (bsAb) designed in the BiTE (bispecific T-cell engager) format, is approved by the US Food and Drug Administration for the treatment of relapsed or refractory B-cell precursor acute lymphoblastic leukemia. Because of its short half-life of 2.1 hours, blinatumomab requires continuous intravenous dosing for efficacy. We developed a novel CD19/CD3 bsAb in the single-chain Fv-Fc format (CD19/CD3-scFv-Fc) with a half-life of â¼5 days. In in vitro experiments, both CD19/CD3-scFv-Fc and blinatumomab induced >90% killing of CLL cells from treatment-naïve patients. Antileukemic activity was associated with increased autologous CD8 and CD4 T-cell proliferation, activation, and granzyme B expression. In the NOD/SCID/IL2Rγnull patient-derived xenograft mouse model, once-weekly treatment with CD19/CD3-scFv-Fc eliminated >98% of treatment-naïve CLL cells in blood and spleen. By contrast, blinatumomab failed to induce a response, even when administered daily. We next explored the activity of CD19/CD3-scFv-Fc in the context of ibrutinib treatment and ibrutinib resistance. CD19/CD3-scFv-Fc induced more rapid killing of CLL cells from ibrutinib-treated patients than those from treatment-naïve 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. Taken together, these data support investigation of CD19/CD3 bsAb's and other T cell-recruiting bsAb's as immunotherapies for CLL, especially in combination with ibrutinib or as rescue therapy in ibrutinib-resistant disease.
Subject(s)
Antibodies, Bispecific/therapeutic use , Antigens, CD19/immunology , CD3 Complex/immunology , Drug Resistance, Neoplasm/drug effects , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Salvage Therapy , Adenine/analogs & derivatives , Animals , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Piperidines , Single-Chain Antibodies/immunology , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
T-cell engaging bispecific antibodies (T-biAbs) mediate potent and selective cytotoxicity by combining specificities for target and effector cells in one molecule. Chemically programmed T-biAbs (cp-T-biAbs) are precisely assembled compositions of (i) small molecules that govern cancer cell surface targeting with high affinity and specificity and (ii) antibodies that recruit and activate T cells and equip the small molecule with confined biodistribution and longer circulatory half-life. Conceptually similar to cp-T-biAbs, switchable chimeric antigen receptor T cells (sCAR-Ts) can also be put under the control of small molecules by using a chemically programmed antibody as a bispecific adaptor molecule. As such, cp-T-biAbs and cp-sCAR-Ts can endow small molecules with the power of cancer immunotherapy. We here review the concept of chemically programmed antibodies for recruiting and activating T cells as a promising strategy for broadening the utility of small molecules in cancer therapy.
Subject(s)
Antibodies, Bispecific/chemistry , Receptors, Chimeric Antigen/chemistry , T-Lymphocytes, Cytotoxic/chemistry , Antibodies, Bispecific/immunology , Humans , Immunotherapy , Immunotherapy, Adoptive , Neoplasms/immunology , Neoplasms/pathology , Neoplasms/therapy , Peptides/chemistry , Receptors, Chimeric Antigen/immunology , Small Molecule Libraries/chemistry , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
Bispecific antibodies (biAb) targeting two different antigens or two distinct epitopes on the same antigen have demonstrated broad therapeutic utility. CD52 and CD20 are co-expressed on the cell surface of malignant B cells in B-cell non-Hodgkin lymphoma (B-NHL) and chronic lymphocytic leukemia (CLL) and increased expression of both antigens is detected on dividing or recently divided cells ("proliferative fraction") in CLL. The CD52-targeting monoclonal antibody (mAb) alemtuzumab (atz) not only depletes malignant B cells but also healthy CD52+ B and T lymphocytes and monocytes, causing severe immunosuppression. Loss of CD20 can occur in CLL after treatment with rituximab (rtx) and other CD20-targeting mAbs. To broaden the benefit of atz and rtx, we engineered an IgG1-like biAb, atzâ¯×â¯rtx scFv-Fc. The Fc fragment of the biAb facilitates purification by Protein A affinity chromatography and supports a longer circulatory half-life. While atzâ¯×â¯rtx scFv-Fc retained both antigen binding specificities, it showed superior binding to CD52+CD20+ B cells compared to CD52+CD20- T cells. Moreover, atzâ¯×â¯rtx scFv-Fc mediated potent complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in vitro and exhibited B-cell depleting but T-cell sparing activities in vivo in a CLL patient-derived xenograft model. B-cell depletion was more pronounced for cells of the proliferative fraction.
Subject(s)
Antibodies, Bispecific/therapeutic use , Antigens, CD20/immunology , CD52 Antigen/antagonists & inhibitors , Immunotherapy , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Alemtuzumab , Animals , Antibodies, Bispecific/pharmacology , Antigens, Neoplasm/immunology , Antineoplastic Agents, Immunological/pharmacology , Antineoplastic Agents, Immunological/therapeutic use , CD52 Antigen/immunology , Humans , Immunoglobulin Fc Fragments , Immunoglobulin G/pharmacology , Immunoglobulin G/therapeutic use , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Mice , Rituximab , Xenograft Model Antitumor AssaysABSTRACT
Although macromolecules on cell surfaces are predominantly targeted and drugged with antibodies, they harbor pockets that are only accessible to small molecules and constitutes a rich subset of binding sites with immense potential diagnostic and therapeutic utility. Compared to antibodies, however, small molecules are disadvantaged by a less confined biodistribution, shorter circulatory half-life, and inability to communicate with the immune system. Presented herein is a method that endows small molecules with the ability to recruit and activate chimeric antigen receptor Tâ cells (CAR-Ts). It is based on a CAR-T platform that uses a chemically programmed antibody fragment (cp-Fab) as on/off switch. In proof-of-concept studies, this cp-Fab/CAR-T system targeting folate binding proteins on the cell surface mediated potent and specific eradication of folate-receptor-expressing cancer cells inâ vitro and inâ vivo.
Subject(s)
Immunotherapy, Adoptive/methods , Receptors, Chimeric Antigen , Animals , Antigens, Neoplasm/immunology , Cell Line, Tumor , Folate Receptor 2 , Humans , Mice , Receptors, Antigen, T-Cell/immunology , Receptors, Cell Surface , Tissue Distribution , Xenograft Model Antitumor AssaysABSTRACT
Chronic lymphocytic leukemia (CLL) is a disease in which a single B-cell clone proliferates relentlessly in peripheral lymphoid organs, bone marrow, and blood. DNA sequencing experiments have shown that about 30% of CLL patients have stereotyped antigen-specific B-cell receptors (BCRs) with a high level of sequence homology in the variable domains of the heavy and light chains. These include many of the most aggressive cases that haveIGHV-unmutated BCRs whose sequences have not diverged significantly from the germ line. This suggests a personalized therapy strategy in which a toxin or immune effector function is delivered selectively to the pathogenic B-cells but not to healthy B-cells. To execute this strategy, serum-stable, drug-like compounds able to target the antigen-binding sites of most or all patients in a stereotyped subset are required. We demonstrate here the feasibility of this approach with the discovery of selective, high affinity ligands for CLL BCRs of the aggressive, stereotyped subset 7P that cross-react with the BCRs of several CLL patients in subset 7p, but not with BCRs from patients outside this subset.
Subject(s)
Antigens/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Receptors, Antigen, B-Cell/immunology , Antigens/genetics , Female , Humans , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Male , Receptors, Antigen, B-Cell/genetics , Tumor Cells, CulturedABSTRACT
Chemically programmed bispecific antibodies (biAbs) endow target cell-binding small molecules with the ability to recruit and activate effector cells of the immune system. Here we report a platform of chemically programmed biAbs aimed at redirecting cytotoxic T cells to eliminate cancer cells. Two different antibody technologies were merged together to make a novel chemically programmed biAb. This was achieved by combining the humanized anti-hapten monoclonal antibody (mAb) h38C2 with the humanized anti-human CD3 mAb v9 in a clinically investigated diabody format known as Dual-Affinity Re-Targeting (DART). We show that h38C2 × v9 DARTs can readily be equipped with tumor-targeting hapten-derivatized small molecules without causing a systemic response harming healthy tissues. As a proof of concept, we chemically programmed h38C2 × v9 with hapten-folate and demonstrated its selectivity and potency against folate receptor 1 (FOLR1)-expressing ovarian cancer cells in vitro and in vivo Unlike conventional biAbs, chemically programmed biAbs in DART format are highly modular with broad utility in terms of both target and effector cell engagement. Most importantly, they provide tumor-targeting compounds access to the power of cancer immunotherapy.
Subject(s)
Antibodies, Bispecific/chemistry , Antibodies, Monoclonal, Humanized/chemistry , Antibodies, Neoplasm/chemistry , Antibodies, Bispecific/immunology , Antibodies, Monoclonal, Humanized/immunology , Antibodies, Neoplasm/immunology , Cell Line, Tumor , Female , Folate Receptor 1/antagonists & inhibitors , Folate Receptor 1/chemistry , Folate Receptor 1/immunology , HEK293 Cells , Humans , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/chemistry , Neoplasm Proteins/immunology , Ovarian Neoplasms/chemistry , Ovarian Neoplasms/immunologyABSTRACT
Control of integrin activation and signaling plays crucial roles in cell adhesion, spreading and migration. Here, we report that selective breakage of two conserved disulfide bonds located at the knees of integrin α4C589-C594 and ß7C494-C526 activated α4ß7. This activated integrin had a unique structure that was different from the typical extended conformation of active integrin. In addition, these activated α4ß7 integrins spontaneously clustered on the cell membrane and triggered integrin downstream signaling independent of ligand binding. Although these disulfide bonds were not broken during α4ß7 activation by inside-out signaling or Mn(2+), they could be specifically reduced by 0.1 mM dithiothreitol, a reducing strength that could be produced in vivo under certain conditions. Our findings reveal a novel mechanism of integrin activation under specific reducing conditions by which integrin can signal and promote cell spreading in the absence of ligand.
Subject(s)
Disulfides/metabolism , Integrin alpha4/chemistry , Integrin alpha4/metabolism , Integrin beta Chains/chemistry , Integrin beta Chains/metabolism , Signal Transduction , Amino Acid Sequence , Humans , Ligands , Molecular Sequence Data , Protein Conformation , Sequence AlignmentABSTRACT
Integrin α(4)ß(7) is a lymphocyte homing receptor that mediates both rolling and firm adhesion of lymphocytes on vascular endothelium, two of the critical steps in lymphocyte migration and tissue-specific homing. The rolling and firm adhesions of lymphocytes rely on the dynamic shift between the inactive and active states of integrin α(4)ß(7), which is associated with the conformational rearrangement of integrin molecules. Activation-specific antibodies, which specifically recognize the activated integrins, have been used as powerful tools in integrin studies, whereas there is no well characterized activation-specific antibody to integrin α(4)ß(7). Here, we report the identification, characterization, and epitope mapping of an activation-specific human mAb J19 against integrin α(4)ß(7). J19 was discovered by screening a human single-chain variable fragment phage library using an activated α(4)ß(7) mutant as target. J19 IgG specifically bound to the high affinity α(4)ß(7) induced by Mn(2+), DTT, ADP, or CXCL12, but not to the low affinity integrin. Moreover, J19 IgG did not interfere with α(4)ß(7)-MAdCAM-1 interaction. The epitope of J19 IgG was mapped to Ser-331, Ala-332, and Ala-333 of ß(7) I domain and a seven-residue segment from 184 to 190 of α(4) ß-propeller domain, which are buried in low affinity integrin with bent conformation and only exposed in the high affinity extended conformation. Taken together, J19 is a potentially powerful tool for both studies on α(4)ß(7) activation mechanism and development of novel therapeutics targeting the activated lymphocyte expressing high affinity α(4)ß(7).
Subject(s)
Antibodies, Monoclonal/immunology , Antibody Specificity/immunology , Epitope Mapping/methods , Integrins/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , Flow Cytometry , HEK293 Cells , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Integrins/chemistry , Integrins/genetics , K562 Cells , Mice , Microscopy, Fluorescence , Models, Molecular , Mutation , Peptide Library , Polysaccharides/chemistry , Polysaccharides/metabolism , Protein Binding , Protein Conformation , Protein Multimerization , Protein Structure, Tertiary , Rats , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunology , Single-Chain Antibodies/metabolismABSTRACT
Integrin α(4)ß(7) mediates rolling and firm adhesion of leucocytes, two of the critical steps in leukocyte migration and tissue specific homing. Affinity of α(4)ß(7) for ligand is dynamically regulated by three interlinked metal ion-binding sites in ß(7)-subunit I domain. In this study, we found that Phe185 (F185), a highly conserved aromatic residue in ß(7)-subunit, links the specificity-determining loop and the synergistic metal ion-binding site (SyMBS) through cation-π interaction. Mutations of F185 that disrupted the SyMBS cation-F185 interaction led to deficient firm cell adhesion mediated by high affinity α(4)ß(7), and only slightly affected rolling adhesion mediated by low affinity α(4)ß(7). Disruption of SyMBS cation-F185 interaction induced partial extension of integrin ectodomain and separation of cytoplasmic tails, and impaired α(4)ß(7)-mediated bidirectional signaling. In addition, loss of SyMBS cation-F185 interaction increased paxillin expression and promoted paxillin-integrin binding, leading to deficient cell spreading. Furthermore, integrin α(4)ß(7)-mediated cell migration was decreased by the abolishment of SyMBS cation-F185 interaction. Thus, these findings reveal a cation-π interaction playing vital roles in the regulation of integrin affinity, signaling, and biological functions.
Subject(s)
Cations/metabolism , Integrins/chemistry , Ligands , Signal Transduction/physiology , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Cricetulus , Humans , Integrins/genetics , Integrins/metabolism , Leukocyte Rolling/physiology , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Sequence AlignmentABSTRACT
Lymphocyte homing is regulated by the dynamic interaction between integrins and their ligands. Integrin α4ß7 mediates both rolling and firm adhesion of lymphocytes by modulating its affinity to the ligand, mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Although previous studies have revealed some mechanisms of α4ß7-MAdCAM-1 binding, little is known about the different molecular bases of the low- and high-affinity α4ß7-MAdCAM-1 interactions, which mediate rolling and firm adhesion of lymphocytes, respectively. Here, we found that two loops in immunoglobulin domains 1 and 2 (D1 and D2) of MAdCAM-1 played different roles in MAdCAM-1 binding to low-affinity (inactive) and high-affinity (activated) α4ß7. The Asp-42 in the CC' loop of D1 was indispensable for MAdCAM-1 binding to both low-affinity and high-affinity α4ß7. The other CC' loop residues except for Arg-39 and Ser-44 were essential for MAdCAM-1 binding to both inactive α4ß7 and α4ß7 activated by SDF-1α or talin, but not required for MAdCAM-1 binding to Mn2+-activated α4ß7. Single amino acid substitution of the DE loop residues mildly decreased MAdCAM-1 binding to both inactive and activated α4ß7. Notably, removal of the DE loop greatly impaired MAdCAM-1 binding to inactive and SDF-1α- or talin-activated α4ß7, but only decreased 60% of MAdCAM-1 binding to Mn2+-activated α4ß7. Moreover, DE loop residues were important for stabilizing the low-affinity α4ß7-MAdCAM-1 interaction. Thus, our findings demonstrate the distinct roles of the CC' and DE loops in the recognition of MAdCAM-1 by low- and high-affinity α4ß7 and suggest that the inactive α4ß7 and α4ß7 activated by different stimuli have distinct conformations with different structural requirements for MAdCAM-1 binding.
Subject(s)
Immunoglobulins/chemistry , Immunoglobulins/metabolism , Integrins/metabolism , Mucoproteins/chemistry , Mucoproteins/metabolism , Cell Adhesion/genetics , Cell Adhesion/physiology , Cell Adhesion Molecules , Cell Line , Humans , Immunoglobulins/genetics , Integrins/genetics , Mucoproteins/genetics , Point Mutation/genetics , Protein Binding/genetics , Protein Binding/physiology , Protein Structure, Secondary , Structure-Activity RelationshipABSTRACT
Change in habitual diet may negatively affect health. The domestic silkworm (Bombyx mori) is an economically important oligophagous insect that feeds on mulberry leaves. The growth, development, and immune-disease resistance of silkworms have declined under artificial dietary conditions. In this study, we used B. mori as a model insect to explore the relationship between changes in diet and balance of intestinal microbes due to its simpler guts compared with those of mammals. We found that artificial diets reduced the intestinal bacterial diversity in silkworms and resulted in a simple intestinal microbial structure. By analyzing the correlations among food, gut, and fecal microbial diversity, we found that an artificial diet was more easily fermented and enriched the lactic acid bacteria in the gut of the silkworms. This diet caused intestinal acidification and microbial imbalance (dysbiosis). When combined with the artificial diet, Enterococcus mundtii, a colonizing opportunistic pathogen, caused dysbiosis and allowed the frequent outbreak of bacterial diseases in the silkworms. This study provides further systematic indicators and technical references for future investigations of the relationship between diet-based environmental changes and intestinal microbial balance. IMPORTANCE The body often appears unwell after habitual dietary changes. The domestic silkworm (Bombyx mori) raised on artificial diets is a good model to explore the relationship between dietary changes and the balance of intestinal microbes. In this study, the food-gut-feces microbial model was established, and some potential key genera that could regulate the balance of intestinal microbiota were screened out. Our findings will provide a reference for future research to further our understanding of healthy silkworm development and may even be useful for similar research on other animals.
Subject(s)
Bombyx , Gastrointestinal Microbiome , Morus , Animals , Bombyx/microbiology , Dysbiosis , Feces , MammalsABSTRACT
BACKGROUND: Despite numerous therapeutic options, safe and curative therapy is unavailable for most patients with chronic lymphocytic leukemia (CLL). A drawback of current therapies such as the anti-CD20 monoclonal antibody (mAb) rituximab is the elimination of all healthy B cells, resulting in impaired humoral immunity. We previously reported the identification of a patient-derived, CLL-binding mAb, JML-1, and identified sialic acid-binding immunoglobulin-like lectin-6 (Siglec-6) as the target of JML-1. Although little is known about Siglec-6, it appears to be an attractive target for cancer immunotherapy due to its absence on most healthy cells and tissues. METHODS: We used a target-specific approach to mine for additional patient-derived anti-Siglec-6 mAbs. To assess the therapeutic utility of targeting Siglec-6 in the context of CLL, T cell-recruiting bispecific antibodies (T-biAbs) that bind to Siglec-6 and CD3 were engineered into single-chain variable fragment-Fc and dual-affinity retargeting (DART)-Fc constructs. T-biAbs were evaluated for their activity in vitro, ex vivo, and in vivo. RESULTS: We discovered the anti-Siglec-6 mAbs RC-1 and RC-2, which bind with higher affinity than JML-1 yet maintain similar specificity. Both JML-1 and RC-1 T-biAbs were effective at activating T cells and killing Siglec-6+ target cells. The RC-1 clone in the DART-Fc format was the most potent T-biAb tested and was the only anti-Siglec-6 T-biAb that eliminated Siglec-6+ primary CLL cells via autologous T cells at pathological T-to-CLL cell ratios. Tested at healthy T-to-B cell ratios, it also eliminated a Siglec-6+ fraction of primary B cells from healthy donors. The subpicomolar potency of the DART-Fc format was attributed to the reduction in the length and flexibility of the cytolytic synapse. Furthermore, the RC-1 T-biAb was effective at clearing MEC1 CLL cells in vivo and demonstrated a circulatory half-life of over 7 days. CONCLUSION: Siglec-6-targeting T-biAbs are highly potent and specific for eliminating Siglec-6+ leukemic and healthy B cells while sparing Siglec-6- healthy B cells, suggesting a unique treatment strategy for CLL with diminished suppression of humoral immunity. Our data corroborate reports that T-biAb efficacy is dependent on synapse geometry and reveal that synapse architecture can be tuned via antibody engineering. Our fully human anti-Siglec-6 antibodies and T-biAbs have potential for cancer immunotherapy. TRIAL REGISTRATION NUMBER: NCT00923507.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , T-Lymphocytes , B-Lymphocytes , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , ImmunotherapyABSTRACT
T-cell engaging bispecific antibodies (biAbs) can mediate potent and specific tumor cell eradication in liquid cancers. Substantial effort has been invested in expanding this concept to solid cancers. To explore their utility in the treatment of ovarian cancer, we built a set of asymmetric biAbs in IgG1-like format that bind CD3 on T cells with a conventional scFv arm and folate receptor 1 (FOLR1) on ovarian cancer cells with a conventional or a chemically programmed Fab arm. For avidity engineering, we also built an asymmetric biAb format with a tandem Fab arm. We show that both conventional and chemically programmed CD3 × FOLR1 biAbs exert specific in vitro and in vivo cytotoxicity toward FOLR1-expressing ovarian cancer cells by recruiting and activating T cells. While the conventional T-cell engaging biAb was curative in an aggressive mouse model of human ovarian cancer, the potency of the chemically programmed biAb was significantly boosted by avidity engineering. Both conventional and chemically programmed CD3 × FOLR1 biAbs warrant further investigation for ovarian cancer immunotherapy.
Subject(s)
Antibodies, Bispecific/pharmacology , Folate Receptor 1/immunology , Ovarian Neoplasms/immunology , Animals , Antibodies, Bispecific/pharmacokinetics , Cell Line , Female , Humans , Lymphocyte Activation , Mice, Inbred BALB C , T-Lymphocytes/immunologyABSTRACT
Owing to their high affinities and specificities, rabbit monoclonal antibodies (mAbs) have demonstrated value and potential primarily as basic research and diagnostic reagents, but, in some cases, also as therapeutics. To accelerate access to rabbit mAbs bypassing immunization, we generated a large naïve rabbit antibody repertoire represented by a phage display library encompassing >10 billion independent antibodies in chimeric rabbit/human Fab format and validated it by next-generation sequencing. Panels of rabbit mAbs selected from this library against two emerging cancer targets, ROR1 and ROR2, revealed high diversity, affinity, and specificity. Moreover, ROR1- and ROR2-targeting rabbit mAbs demonstrated therapeutic utility as components of chimeric antigen receptor-engineered T cells, further corroborating the value of the naïve rabbit antibody library as a rich and virtually unlimited source of rabbit mAbs.
Subject(s)
Antibodies, Monoclonal/isolation & purification , Immunologic Factors/isolation & purification , Animals , Antibodies, Monoclonal/therapeutic use , Antigens, Neoplasm/immunology , Cell Line, Tumor , Cell Proliferation , Cytokines/metabolism , Cytotoxicity Tests, Immunologic , Humans , Immunoglobulin Fab Fragments/genetics , Immunologic Factors/therapeutic use , Immunotherapy/methods , Peptide Library , Rabbits , Receptor Tyrosine Kinase-like Orphan Receptors/immunology , Recombinant Proteins/isolation & purification , Recombinant Proteins/therapeutic use , T-Lymphocytes/immunologyABSTRACT
AIM OF THE STUDY: To examine the antiproliferation and anti-invasion of Eupolyphaga sinensis Walker 70% ethanol extract (ESWE) on breast cancer and elucidate the underlying signaling mechanisms. METHODS: MTT and colony formation assays were used to investigate the effect of ESWE on proliferation of breast cancer cells in vitro. The xenograft mouse tumor model was used to determine the effect of ESWE on breast cancer in vivo. To investigate the underlying molecular mechanisms, we used western blotting to analyze the expression of ERK1/2, CXCR4, matrix metalloproteinase 2 (MMP2), and MMP9 pretreated with ESWE. The stromal cell-derived factor (SDF)-1α-induced migration and invasion potential of breast cancer cells were examined by wound-healing assays and Matrigel invasion chamber assays. RESULTS: ESWE effectively inhibited the proliferation of MDA-MB-435s and MDA-MB-231 cells and exhibited antitumor effects in an MDA-MB-231 xenograft mice model. Furthermore, ESWE suppressed the activity of ERK1/2, a key molecule of MAPK signaling. We also observed that ESWE treatment led to downregulation of CXCR4 expression as well as greatly reduced MMP2 and MMP9. ESWE affected CXCR4 expression partially through the modulation of autocrine vascular endothelial growth factor. However, suppression of CXCR4 expression was the result of downregulation of mRNA expression. Inhibition of CXCR4 expression by ESWE further correlated with the suppression of SDF-1α-induced migration and invasion in breast cancer cells. CONCLUSION: ESWE exerted its antiproliferation and antiinvasion by regulating MAPK signaling and related metastasis factorsand thus could be a useful therapeutic candidate for breast cancer intervention.
Subject(s)
Biological Factors/pharmacology , Breast Neoplasms/drug therapy , Cell Movement/drug effects , Cell Proliferation/drug effects , Cockroaches/chemistry , Ethanol/chemistry , Neoplasm Invasiveness/prevention & control , Animals , Breast Neoplasms/metabolism , Cell Line, Tumor , Female , Humans , MAP Kinase Signaling System/drug effects , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Inbred BALB C , Receptors, CXCR4/metabolism , Signal Transduction/drug effects , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Cancer cell growth is complicated progression which is regulated and controlled by multiple factors including cell cycle, migration and apoptosis. In present study, we report that TADs, a novel derivative of taspine, has an essential role in resisting hepatocellular carcinoma growth (including arrest cell cycle) and migration, and inducing cell apoptosis. Our findings demonstrated that the TADs showed good inhibition on the hepatoma cell growth and migration, and good action on apoptosis induction. Using genome-wide microarray analysis, we found the down-regulated growth and apoptosis factors, and selected down-regulated genes were confirmed by Western blot. Knockdown of a checkpoint c-Myc by siRNA significantly attenuated tumor inhibition and apoptosis effects of TADs. Moreover, our results indicated TADs could simultaneously increase cyclin D1 protein levels and decrease amount of cyclin E, cyclin B1 and cdc2 of the cycle proteins, and also TADs reduced Bcl-2 expression, and upregulated Bad, Bak and Bax activities. In conclusion, these results illustrated that TADs is a key factor in growth and apoptosis signaling inhibitor, has potential in cancer therapy.