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1.
Blood ; 129(5): 609-618, 2017 02 02.
Article in English | MEDLINE | ID: mdl-27908880

ABSTRACT

Acute myeloid leukemia (AML) is a major unmet medical need. Most patients have poor long-term survival, and treatment has not significantly changed in 40 years. Recently, bispecific antibodies that redirect the cytotoxic activity of effector T cells by binding to CD3, the signaling component of the T-cell receptor, and a tumor target have shown clinical activity. Notably, blinatumomab is approved to treat relapsed/refractory acute lymphoid leukemia. Here we describe the design, discovery, pharmacologic activity, pharmacokinetics, and safety of a CD3 T cell-dependent bispecific (TDB) full-length human IgG1 therapeutic antibody targeting CLL-1 that could potentially be used in humans to treat AML. CLL-1 is prevalent in AML and, unlike other targets such as CD33 and CD123, is not expressed on hematopoietic stem cells providing potential hematopoietic recovery. We selected a high-affinity monkey cross-reactive anti-CLL-1 arm and tested several anti-CD3 arms that varied in affinity, and determined that the high-affinity CD3 arms were up to 100-fold more potent in vitro. However, in mouse models, the efficacy differences were less pronounced, probably because of prolonged exposure to TDB found with lower-affinity CD3 TDBs. In monkeys, assessment of safety and target cell depletion by the high- and low-affinity TDBs revealed that only the low-affinity CD3/CLL1 TDB was well tolerated and able to deplete target cells. Our data suggest that an appropriately engineered CLL-1 TDB could be effective in the treatment of AML.


Subject(s)
Antibodies, Bispecific/therapeutic use , Antineoplastic Agents/therapeutic use , Lectins, C-Type/immunology , Leukemia, Myeloid, Acute/drug therapy , Sialic Acid Binding Ig-like Lectin 3/immunology , Animals , Antibodies, Bispecific/adverse effects , Antibodies, Bispecific/immunology , Antibodies, Bispecific/pharmacokinetics , Antineoplastic Agents/adverse effects , Antineoplastic Agents/immunology , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Humans , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/pathology , Macaca fascicularis , Mice, Inbred C57BL , Mice, Transgenic
2.
Chemistry ; 24(19): 4830-4834, 2018 Apr 03.
Article in English | MEDLINE | ID: mdl-29493023

ABSTRACT

A novel strategy to attach indole-containing payloads to antibodies through a carbamate moiety and a self-immolating, disulfide-based linker is described. This new strategy was employed to connect a selective estrogen receptor down-regulator (SERD) to various antibodies in a site-selective manner. The resulting conjugates displayed potent, antigen-dependent down-regulation of estrogen receptor levels in MCF7-neo/HER2 and MCF7-hB7H4 cells. They also exhibited similar antigen-dependent modulation of the estrogen receptor in tumors when administered intravenously to mice bearing MCF7-neo/HER2 tumor xenografts. The indole-carbamate moiety present in the new linker was stable in whole blood from various species and also exhibited good inĆ¢Ā€Ā…vivo stability properties in mice.


Subject(s)
Indoles/chemistry , Animals , Antibodies, Monoclonal/chemistry , Cell Line, Tumor , Humans , Immunoconjugates/administration & dosage , MCF-7 Cells , Mice
3.
Mol Pharm ; 12(6): 1717-29, 2015 Jun 01.
Article in English | MEDLINE | ID: mdl-25853436

ABSTRACT

B7-H4 has been implicated in cancers of the female reproductive system and investigated for its possible use as a biomarker for cancer, but there are no preclinical studies to demonstrate that B7-H4 is a molecular target for therapeutic intervention of cancer. We provide evidence that the prevalence and expression levels of B7-H4 are high in different subtypes of breast cancer and that only a few normal tissues express B7-H4 on the cell membrane. These profiles of low normal expression and upregulation in cancer provide an opportunity for the use of antibody-drug conjugates (ADCs), cytotoxic drugs chemically linked to antibodies, for the treatment of B7-H4 positive cancers. We have developed an ADC specific to B7-H4 that uses a linker drug consisting of a potent antimitotic, monomethyl auristatin E (MMAE), linked to engineered cysteines (THIOMAB) via a protease labile linker. We will refer to ADCs that use the THIOMAB format as TDCs to help distinguish the format from standard MC-vc-MMAE ADCs that are conjugated to the interchain disulfide bonds. Anti-B7-H4 (h1D11)-MC-vc-PAB-MMAE (h1D11 TDC) produced durable tumor regression in cell line and patient-derived xenograft models of triple-negative breast cancer. It also binds rat B7-H4 with similar affinity to human and allowed us to test for target dependent toxicity in rats. We found that our anti-B7-H4 TDC has toxicity findings similar to untargeted TDC. Our results validate B7-H4 as an ADC target for breast cancer and support the possible use of this TDC in the treatment of B7-H4(+) breast cancer.


Subject(s)
Antineoplastic Agents/therapeutic use , Immunoconjugates/therapeutic use , Oligopeptides/therapeutic use , Animals , Antineoplastic Agents/chemistry , Blotting, Western , Cell Line, Tumor , Female , Flow Cytometry , Humans , Immunoconjugates/chemistry , Immunohistochemistry , Mice , Mice, SCID , Oligopeptides/chemistry , Rats , Rats, Sprague-Dawley , Triple Negative Breast Neoplasms/drug therapy , Xenograft Model Antitumor Assays
4.
MAbs ; 14(1): 2115213, 2022.
Article in English | MEDLINE | ID: mdl-36206404

ABSTRACT

T cell-engaging bispecific antibodies (TCEs) are clinically effective treatments for hematological cancers. While the utility of TCEs in solid malignancies is being explored, toxicities arising from antigen expression on normal tissues have slowed or halted several clinical trials. Here, we describe the development of TCEs that preferentially drive T cell-mediated death against target cells co-expressing two tumor-associated antigens. We show that Ly6E and B7-H4 are simultaneously expressed on approximately 50% of breast cancers, whereas normal tissue expression is limited and mostly orthogonal. Traditional bispecific TCEs targeting a singular antigen, either Ly6E or B7-H4, are active when paired with high-affinity CD3-engagers, but normal tissue expression presents a toxicity risk. Treatment with a murine cross-reactive B7-H4-TCE results in rapid and severe weight loss in mice along with damage to B7-H4-expressing tissues. To overcome on-target toxicity, we designed trispecific antibodies co-targeting Ly6E, B7-H4, and CD3 and characterized the impact of dual-antigen binding and the relative placement of each binding domain on tumor killing in vitro and in vivo. In vitro killing of tumor cells co-expressing both antigens correlates to the placement of the higher affinity B7-H4 binding domain, with only modest enhancements seen upon addition of Ly6E binding. In xenograft models, avid binding of appropriately designed trispecific TCEs enables tumor growth inhibition while evading the poor tolerability seen with active bispecific TCEs. Collectively these data highlight the potential for dual-antigen targeting to improve safety and efficacy, and expand the scope of tumors that may effectively be treated by TCEs.Abbreviations: Chimeric antigen receptor T cells (CAR-Ts), dual-antigen targeted T cell engagers (DAT-TCE), Fragment antigen-binding (Fab), Hematoxylin and eosin (H&E), Institutional Animal Care and Use Committee (IACUC), Immunoglobulin G (IgG), immunohistochemistry (IHC), NOD SCID gamma (NSG), peripheral blood mononuclear cells (PBMCs), surface plasmon resonance (SPR), T cell-engagers (TCEs).


Subject(s)
Antibodies, Bispecific , Receptors, Chimeric Antigen , Animals , Antibodies, Bispecific/pharmacology , Cell Line, Tumor , Eosine Yellowish-(YS) , Hematoxylin , Humans , Immunoglobulin G , Leukocytes, Mononuclear , Mice , Mice, SCID , T-Lymphocytes , Xenograft Model Antitumor Assays
5.
Mol Cancer Ther ; 21(6): 974-985, 2022 06 01.
Article in English | MEDLINE | ID: mdl-35364611

ABSTRACT

New therapeutics and combination regimens have led to marked clinical improvements for the treatment of a subset of colorectal cancer. Immune checkpoint inhibitors have shown clinical efficacy in patients with mismatch-repair-deficient or microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC). However, patients with microsatellite-stable (MSS) or low levels of microsatellite instable (MSI-L) colorectal cancer have not benefited from these immune modulators, and the survival outcome remains poor for the majority of patients diagnosed with mCRC. In this article, we describe the discovery of a novel T-cell-dependent bispecific antibody (TDB) targeting tumor-associated antigen LY6G6D, LY6G6D-TDB, for the treatment of colorectal cancer. RNAseq analysis showed that LY6G6D was differentially expressed in colorectal cancer with high prevalence in MSS and MSI-L subsets, whereas LY6G6D expression in normal tissues was limited. IHC confirmed the elevated expression of LY6G6D in primary and metastatic colorectal tumors, whereas minimal or no expression was observed in most normal tissue samples. The optimized LY6G6D-TDB, which targets a membrane-proximal epitope of LY6G6D and binds to CD3 with high affinity, exhibits potent antitumor activity both in vitro and in vivo. In vitro functional assays show that LY6G6D-TDB-mediated T-cell activation and cytotoxicity are conditional and target dependent. In mouse xenograft tumor models, LY6G6D-TDB demonstrates antitumor efficacy as a single agent against established colorectal tumors, and enhanced efficacy can be achieved when LY6G6D-TDB is combined with PD-1 blockade. Our studies provide evidence for the therapeutic potential of LY6G6D-TDB as an effective treatment option for patients with colorectal cancer.


Subject(s)
Antibodies, Bispecific , Colorectal Neoplasms , Immunoglobulins , Animals , Antibodies, Bispecific/immunology , Antibodies, Bispecific/pharmacology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Humans , Immune Checkpoint Inhibitors/pharmacology , Immunoglobulins/immunology , Mice , Microsatellite Instability , T-Lymphocytes/immunology
6.
Leukemia ; 36(4): 1006-1014, 2022 04.
Article in English | MEDLINE | ID: mdl-35001074

ABSTRACT

Despite the recent progress, multiple myeloma (MM) is still essentially incurable and there is a need for additional effective treatments with good tolerability. RO7297089 is a novel bispecific BCMA/CD16A-directed innate cell engager (ICEĀ®) designed to induce BCMA+ MM cell lysis through high affinity binding of CD16A and retargeting of NK cell cytotoxicity and macrophage phagocytosis. Unlike conventional antibodies approved in MM, RO7297089 selectively targets CD16A with no binding of other FcƎĀ³ receptors, including CD16B on neutrophils, and irrespective of 158V/F polymorphism, and its activity is less affected by competing IgG suggesting activity in the presence of M-protein. Structural analysis revealed this is due to selective interaction with a single residue (Y140) uniquely present in CD16A opposite the Fc binding site. RO7297089Ā induced tumor cell killing more potently than conventional antibodies (wild-type and Fc-enhanced) and induced lysis of BCMA+ cells at very low effector-to-target ratios. Preclinical toxicology data suggested a favorable safety profile as in vitro cytokine release was minimal and no RO7297089-related mortalities or adverse events were observed in cynomolgus monkeys. These data suggest good tolerability and the potential of RO7297089 to be a novel effective treatment of MM patients.


Subject(s)
Antibodies, Bispecific , Multiple Myeloma , B-Cell Maturation Antigen , Humans , Multiple Myeloma/drug therapy , Phagocytosis , Receptors, IgG
7.
J Biol Chem ; 285(27): 20850-9, 2010 Jul 02.
Article in English | MEDLINE | ID: mdl-20444694

ABSTRACT

The development of bispecific antibodies as therapeutic agents for human diseases has great clinical potential, but broad application has been hindered by the difficulty of identifying bispecific antibody formats that exhibit favorable pharmacokinetic properties and ease of large-scale manufacturing. Previously, the development of an antibody technology utilizing heavy chain knobs-into-holes mutations and a single common light chain enabled the small-scale generation of human full-length bispecific antibodies. Here we have extended the technology by developing a two-part bispecific antibody discovery strategy that facilitates proof-of-concept studies and clinical candidate antibody generation. Our scheme consists of the efficient small-scale generation of bispecific antibodies lacking a common light chain and the hinge disulfides for proof-of-concept studies coupled with the identification of a common light chain bispecific antibody for large-scale production with high purity and yield. We have applied this technology to generate a bispecific antibody suitable for development as a human therapeutic. This antibody directly inhibits the activation of the high affinity IgE receptor FcepsilonRI on mast cells and basophils by cross-linking FcepsilonRI with the inhibitory receptor FcgammaRIIb, an approach that has strong therapeutic potential for asthma and other allergic diseases. Our approach for producing human bispecific full-length antibodies enables the clinical application of bispecific antibodies to a validated therapeutic pathway in asthma.


Subject(s)
Antibodies, Bispecific/therapeutic use , Receptors, IgE/physiology , Amino Acid Substitution , Animals , Antibodies, Bispecific/genetics , Antibody Specificity , Basophils/immunology , Cell Line, Tumor , Codon/genetics , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Genes , Glutathione Transferase/genetics , Humans , Immunoglobulin E/genetics , Immunoglobulin E/immunology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Mice , Mice, SCID , Passive Cutaneous Anaphylaxis/immunology , Receptors, IgE/antagonists & inhibitors , Receptors, IgE/drug effects , Receptors, IgE/immunology , Receptors, IgG/immunology , Recombinant Proteins/therapeutic use , Retinal Neoplasms/immunology , Retinoblastoma/immunology , Sensitivity and Specificity
8.
Clin Cancer Res ; 25(4): 1358-1368, 2019 02 15.
Article in English | MEDLINE | ID: mdl-29959143

ABSTRACT

PURPOSE: The treatment of acute myeloid leukemia (AML) has not significantly changed in 40 years. Cytarabine- and anthracycline-based chemotherapy induction regimens (7 + 3) remain the standard of care, and most patients have poor long-term survival. The reapproval of Mylotarg, an anti-CD33-calicheamicin antibody-drug conjugate (ADC), has demonstrated ADCs as a clinically validated option to enhance the effectiveness of induction therapy. We are interested in developing a next-generation ADC for AML to improve upon the initial success of Mylotarg. EXPERIMENTAL DESIGN: The expression pattern of CLL-1 and its hematopoietic potential were investigated. A novel anti-CLL-1-ADC, with a highly potent pyrrolobenzodiazepine (PBD) dimer conjugated through a self-immolative disulfide linker, was developed. The efficacy and safety profiles of this ADC were evaluated in mouse xenograft models and in cynomolgus monkeys. RESULTS: We demonstrate that CLL-1 shares similar prevalence and trafficking properties that make CD33 an excellent ADC target for AML, but lacks expression on hematopoietic stem cells that hampers current CD33-targeted ADCs. Our anti-CLL-1-ADC is highly effective at depleting tumor cells in AML xenograft models and lacks target independent toxicities at doses that depleted target monocytes and neutrophils in cynomolgus monkeys. CONCLUSIONS: Collectively, our data suggest that an anti-CLL-1-ADC has the potential to become an effective and safer treatment for AML in humans, by reducing and allowing for faster recovery from initial cytopenias than the current generation of ADCs for AML.


Subject(s)
Antibodies, Anti-Idiotypic/pharmacology , Immunoconjugates/pharmacology , Lectins, C-Type/immunology , Leukemia, Myeloid, Acute/drug therapy , Receptors, Mitogen/immunology , Animals , Flow Cytometry , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lectins, C-Type/antagonists & inhibitors , Lectins, C-Type/genetics , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/pathology , Mice , Receptors, Mitogen/antagonists & inhibitors , Receptors, Mitogen/genetics , Sialic Acid Binding Ig-like Lectin 3/genetics , Sialic Acid Binding Ig-like Lectin 3/immunology , Xenograft Model Antitumor Assays
9.
J Clin Invest ; 120(6): 2218-29, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20458139

ABSTRACT

IgE-mediated hypersensitivity is central to the pathogenesis of asthma and other allergic diseases. Although neutralization of serum IgE with IgE-specific antibodies is in general an efficacious treatment for allergic asthma, one limitation of this approach is its lack of effect on IgE production. Here, we have developed a strategy to disrupt IgE production by generating monoclonal antibodies that target a segment of membrane IgE on human IgE-switched B cells that is not present in serum IgE. This segment is known as the M1' domain, and using genetically modified mice that contain the human M1' domain inserted into the mouse IgE locus, we demonstrated that M1'-specific antibodies reduced serum IgE and IgE-producing plasma cells in vivo, without affecting other immunoglobulin isotypes. M1'-specific antibodies were effective when delivered prophylactically and therapeutically in mouse models of immunization, allergic asthma, and Nippostrongylus brasiliensis infection, likely by inducing apoptosis of IgE-producing B cells. In addition, we generated a humanized M1'-specific antibody that was active on primary human cells in vivo, as determined by its reduction of serum IgE levels and IgE plasma cell numbers in a human PBMC-SCID mouse model. Thus, targeting of human IgE-producing B cells with apoptosis-inducing M1'-specific antibodies may be a novel treatment for asthma and allergy.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies/immunology , B-Lymphocytes/drug effects , Hypersensitivity, Immediate/immunology , Mice, SCID/immunology , Animals , Antibodies, Monoclonal/immunology , Asthma/immunology , B-Lymphocytes/immunology , Humans , Hypersensitivity/immunology , Immunization , Mice , Mice, Transgenic , Nippostrongylus/drug effects , Nippostrongylus/immunology
10.
J Biol Chem ; 277(41): 38660-8, 2002 Oct 11.
Article in English | MEDLINE | ID: mdl-12167647

ABSTRACT

CD28 and CTLA-4 (CD152) play a pivotal role in the regulation of T cell activation. Upon ligation by CD80 (B7-1) or CD86 (B7-2), CD28 induces T cell proliferation, cytokine production, and effector functions, whereas CTLA-4 signaling inhibits expansion of activated T cells and induces tolerance. Therefore, we hypothesized that co-stimulatory molecules that preferentially bind CD28 or CTLA-4 would have dramatically altered biological properties. We describe directed molecular evolution of CD80 genes derived from human, orangutan, rhesus monkey, baboon, cat, cow, and rabbit by DNA shuffling and screening. In contrast to wild-type CD80, the evolved co-stimulatory molecules, termed CD28-binding protein (CD28BP) and CTLA-4-binding protein (CTLA-4BP), selectively bind to CD28 or CTLA-4, respectively. Furthermore, CD28BP has improved capacity to induce human T cell proliferation and interferon-gamma production compared with wild-type CD80. In contrast, CTLA-4BP inhibited human mixed leukocyte reaction (MLR) and enhanced interleukin 10 production in MLR, supporting a role for CTLA-4BP in inducing T cell anergy and tolerance. In addition, co-stimulation of purified human T cells was significantly suppressed when CTLA-4BP was cotransfected with either CD80 or CD28BP. The amino acid sequences of CD28BP and CTLA-4BP were 61 and 96% identical with that of human CD80 and provide insight into the residues that are critical in the ligand binding. These molecules provide a new approach to characterization of CD28 and CTLA-4 signals and to manipulation of the T cell response.


Subject(s)
Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , CD28 Antigens/genetics , CD28 Antigens/metabolism , Carrier Proteins/metabolism , Immunoconjugates , Recombinant Fusion Proteins/metabolism , Recombinant Proteins , T-Lymphocytes/metabolism , Abatacept , Amino Acid Sequence , Animals , Antigens, CD , Antigens, Differentiation/immunology , B7-1 Antigen/genetics , B7-1 Antigen/metabolism , CD28 Antigens/immunology , CTLA-4 Antigen , Carrier Proteins/genetics , Carrier Proteins/immunology , Cell Line , Cytokines/metabolism , DNA Shuffling , DNA, Complementary/genetics , DNA, Complementary/metabolism , Evolution, Molecular , Flow Cytometry , Humans , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/immunology , Immunoglobulin Fc Fragments/metabolism , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/radiation effects , Ligands , Molecular Sequence Data , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Sequence Alignment , T-Lymphocytes/immunology
11.
Proc Natl Acad Sci U S A ; 100(3): 1163-8, 2003 Feb 04.
Article in English | MEDLINE | ID: mdl-12529500

ABSTRACT

DNA delivery of IL-12 has shown promise in reducing the toxic side effects associated with administration of recombinant human (h)IL-12 protein while maintaining the ability to inhibit tumor growth and abolish tumor metastases in animal models. We have developed a more potent version of IL-12 by using DNA shuffling and screening to improve its expression in human cells and specific activity on human T cells. The most improved evolved IL-12 (EvIL-12) derived from seven mammalian genes encoding both the p35 and p40 subunits of IL-12 showed a 128-fold improvement in human T cell proliferation compared with native hIL-12 during the initial screening of supernatants from transected cells. When purified hIL-12 and EvIL-12 proteins were compared in vitro in human T cell proliferation and Th1 differentiation assays, it was demonstrated that EvIL-12 exhibited a concomitant 10-fold increase in the specific activity of the protein compared with hIL-12. Furthermore, DNA shuffling improved the level of expression and homogeneity of the heterodimer synthesized by 293 human embryonic kidney cells transfected with EvIL-12 by at least 10-fold. Molecular analysis of the variant revealed strategic placement of amino acid substitutions that potentially may facilitate heterodimer formation and product expression. The enhanced expression and biological activity of EvIL-12 may improve the effectiveness of IL-12 gene-based vaccines and therapeutics without the toxic side effects sometimes associated with hIL-12 protein administration.


Subject(s)
DNA/analysis , Genetic Techniques , Interleukin-12/biosynthesis , Interleukin-12/genetics , Amino Acid Sequence , Animals , COS Cells , Cell Differentiation , Cell Division , Cell Line , Cells, Cultured , Dimerization , Directed Molecular Evolution , Dose-Response Relationship, Drug , Gene Library , Glycosylation , Humans , Models, Molecular , Molecular Sequence Data , T-Lymphocytes , Th1 Cells , Transfection
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