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1.
Clin Transl Sci ; 15(4): 954-966, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34997701

RESUMO

Odronextamab is a fully-human IgG4-based CD20xCD3 bispecific antibody that binds to CD3 on T cells and CD20 on B cells, triggering T-cell-mediated cytotoxicity independent of T-cell-receptor recognition. Adequate safety, tolerability, and encouraging durable complete responses have been observed in an ongoing first-in-human (FIH) study of odronextamab in patients with relapsed/refractory (R/R) B-cell non-Hodgkin lymphoma (B-NHL; NCT02290951). We retrospectively evaluated the pharmacokinetic, pharmacodynamic, and antitumor characteristics of odronextamab in a series of in vitro/in vivo preclinical experiments, to assess their translational value to inform dose escalation for the FIH study. Half-maximal effective concentration values from in vitro cytokine release assays (range: 0.05-0.08 mg/L) provided a reasonable estimate of odronextamab concentrations in patients associated with cytokine release at a 0.5 mg dose (maximum serum concentration: 0.081 mg/L) on week 1/day 1, which could therefore be used to determine the week 1 clinical dose. Odronextamab concentrations resulting in 100% inhibition of tumor growth in a Raji xenograft tumor mouse model (1-10 mg/L) were useful to predict efficacious concentrations in patients and inform dose-escalation strategy. Although predicted human pharmacokinetic parameters derived from monkey data overestimated projected odronextamab exposure, they provided a conservative estimate for FIH starting doses. With step-up dosing, the highest-tested weekly odronextamab dose in patients (320 mg) exceeded the 1 mg/kg single dose in monkeys without step-up dosing. In conclusion, combination of odronextamab in vitro cytokine data, efficacious concentration data from mouse tumor models, and pharmacokinetic evaluations in monkeys has translational value to inform odronextamab FIH study design in patients with R/R B-NHL.


Assuntos
Anticorpos Biespecíficos , Antineoplásicos , Linfoma de Células B , Animais , Antígenos CD20 , Antineoplásicos/uso terapêutico , Citocinas , Humanos , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/patologia , Camundongos , Estudos Retrospectivos
2.
Sci Rep ; 11(1): 14397, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257348

RESUMO

T-cell-redirecting bispecific antibodies have emerged as a new class of therapeutic agents designed to simultaneously bind to T cells via CD3 and to tumor cells via tumor-cell-specific antigens (TSA), inducing T-cell-mediated killing of tumor cells. The promising preclinical and clinical efficacy of TSAxCD3 antibodies is often accompanied by toxicities such as cytokine release syndrome due to T-cell activation. How the efficacy and toxicity profile of the TSAxCD3 bispecific antibodies depends on the binding affinity to CD3 remains unclear. Here, we evaluate bispecific antibodies that were engineered to have a range of CD3 affinities, while retaining the same binding affinity for the selected tumor antigen. These agents were tested for their ability to kill tumor cells in vitro, and their biodistribution, serum half-life, and anti-tumor activity in vivo. Remarkably, by altering the binding affinity for CD3 alone, we can generate bispecific antibodies that maintain potent killing of TSA + tumor cells but display differential patterns of cytokine release, pharmacokinetics, and biodistribution. Therefore, tuning CD3 affinity is a promising method to improve the therapeutic index of T-cell-engaging bispecific antibodies.


Assuntos
Anticorpos Biespecíficos , Complexo CD3 , Citocinas , Citocinas/metabolismo , Ativação Linfocitária , Distribuição Tecidual
3.
Sci Transl Med ; 12(549)2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32581132

RESUMO

Monoclonal antibodies that block the programmed cell death 1 (PD-1) checkpoint have revolutionized cancer immunotherapy. However, many major tumor types remain unresponsive to anti-PD-1 therapy, and even among responsive tumor types, most of the patients do not develop durable antitumor immunity. It has been shown that bispecific antibodies activate T cells by cross-linking the TCR/CD3 complex with a tumor-specific antigen (TSA). The class of TSAxCD3 bispecific antibodies have generated exciting results in early clinical trials. We have recently described another class of "costimulatory bispecifics" that cross-link a TSA to CD28 (TSAxCD28) and cooperate with TSAxCD3 bispecifics. Here, we demonstrate that these TSAxCD28 bispecifics (one specific for prostate cancer and the other for epithelial tumors) can also synergize with the broader anti-PD-1 approach and endow responsiveness-as well as long-term immune memory-against tumors that otherwise do not respond to anti-PD-1 alone. Unlike CD28 superagonists, which broadly activate T cells and induce cytokine storm, TSAxCD28 bispecifics display little or no toxicity when used alone or in combination with a PD-1 blocker in genetically humanized immunocompetent mouse models or in primates and thus may provide a well-tolerated and "off the shelf" combination approach with PD-1 immunotherapy that can markedly enhance antitumor efficacy.


Assuntos
Anticorpos Biespecíficos , Neoplasias , Animais , Anticorpos Biespecíficos/uso terapêutico , Antígenos CD28 , Humanos , Imunoterapia , Camundongos , Neoplasias/tratamento farmacológico , Receptor de Morte Celular Programada 1
4.
Cancer Immunol Res ; 8(5): 596-608, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32184296

RESUMO

Patients with hematologic cancers have improved outcomes after treatment with bispecific antibodies that bind to CD3 on T cells and that redirect T cells toward cancer cells. However, clinical benefit against solid tumors remains to be shown. We made a bispecific antibody that targets both the common prostate tumor-specific antigen PSMA and CD3 (PMSAxCD3) and provide evidence for tumor inhibition in several preclinical solid tumor models. Mice expressing the human extracellular regions of CD3 and PSMA were generated to examine antitumor efficacy in the presence of an intact immune system and PSMA expression in normal tissues. PSMAxCD3 accumulated in PSMA-expressing tissues and tumors as detected by immuno-PET imaging. Although PSMAxCD3 induced T-cell activation and showed antitumor efficacy in mice with low tumor burden, PSMAxCD3 lost efficacy against larger solid tumors, mirroring the difficulty of treating solid tumors in the clinic. Costimulatory receptors can enhance T-cell responses. We show here that costimulation can enhance the antitumor efficacy of PSMAxCD3. In particular, 4-1BB stimulation in combination with PSMAxCD3 enhanced T-cell activation and proliferation, boosted efficacy against larger tumors, and induced T-cell memory, leading to durable antitumor responses. The combination of CD3 bispecific antibodies and anti-4-1BB costimulation represents a therapeutic approach for the treatment of solid tumors.


Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais/farmacologia , Complexo CD3/imunologia , Glutamato Carboxipeptidase II/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Linfócitos T/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Antígenos de Superfície/imunologia , Antígenos de Superfície/metabolismo , Complexo CD3/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Glutamato Carboxipeptidase II/imunologia , Glutamato Carboxipeptidase II/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Radioisótopos/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Zircônio/farmacocinética
5.
Sci Transl Med ; 12(525)2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31915305

RESUMO

T cell activation is initiated upon binding of the T cell receptor (TCR)/CD3 complex to peptide-major histocompatibility complexes ("signal 1"); activation is enhanced by engagement of a second "costimulatory" receptor, such as the CD28 receptor on T cells binding to its cognate ligand(s) on the target cell ("signal 2"). CD3-based bispecific antibodies act by replacing conventional signal 1, linking T cells to tumor cells by binding a tumor-specific antigen (TSA) with one arm of the bispecific and bridging to TCR/CD3 with the other. Although some of these so-called TSAxCD3 bispecifics have demonstrated promising antitumor efficacy in patients with cancer, their activity remains to be optimized. Here, we introduce a class of bispecific antibodies that mimic signal 2 by bridging TSA to the costimulatory CD28 receptor on T cells. We term these TSAxCD28 bispecifics and describe two such bispecific antibodies: one specific for ovarian and the other for prostate cancer antigens. Unlike CD28 superagonists, which broadly activate T cells and resulted in profound toxicity in early clinical trials, these TSAxCD28 bispecifics show limited activity and no toxicity when used alone in genetically humanized immunocompetent mouse models or in primates. However, when combined with TSAxCD3 bispecifics, they enhance the artificial synapse between a T cell and its target cell, potentiate T cell activation, and markedly improve antitumor activity of CD3 bispecifics in a variety of xenogeneic and syngeneic tumor models. Combining this class of CD28-costimulatory bispecific antibodies with the emerging class of TSAxCD3 bispecifics may provide well-tolerated, off-the-shelf antibody therapies with robust antitumor efficacy.


Assuntos
Anticorpos Biespecíficos/imunologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Neoplasias/imunologia , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/metabolismo , Citotoxicidade Imunológica , Feminino , Células HEK293 , Humanos , Sinapses Imunológicas/metabolismo , Ativação Linfocitária/imunologia , Macaca fascicularis , Camundongos , Neoplasias/patologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Sci Transl Med ; 11(497)2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-31217340

RESUMO

Advanced ovarian cancer is frequently treated with combination chemotherapy, but high recurrence rates show the need for therapies that can produce durable responses and extend overall survival. Bispecific antibodies that interact with tumor antigens on cancer cells and activating receptors on immune cells offer an innovative immunotherapy approach. Here, we describe a human bispecific antibody (REGN4018) that binds both Mucin 16 (MUC16), a glycoprotein that is highly expressed on ovarian cancer cells, and CD3, thus bridging MUC16-expressing cells with CD3+ T cells. REGN4018 induced T cell activation and killing of MUC16-expressing tumor cells in vitro. Binding and cytotoxicity of REGN4018 in vitro were minimally affected by high concentrations of CA-125, the shed form of MUC16, which is present in patients. In preclinical studies with human ovarian cancer cells and human T cells in immunodeficient mice, REGN4018 potently inhibited growth of intraperitoneal ovarian tumors. Moreover, in a genetically engineered immunocompetent mouse expressing human CD3 and human MUC16 [humanized target (HuT) mice], REGN4018 inhibited growth of murine tumors expressing human MUC16, and combination with an anti-PD-1 antibody enhanced this efficacy. Immuno-PET imaging demonstrated localization of REGN4018 in MUC16-expressing tumors and in T cell-rich organs such as the spleen and lymph nodes. Toxicology studies in cynomolgus monkeys showed minimal and transient increases in serum cytokines and C-reactive protein after REGN4018 administration, with no overt toxicity. Collectively, these data demonstrate potent antitumor activity and good tolerability of REGN4018, supporting clinical evaluation of REGN4018 in patients with MUC16-expressing advanced ovarian cancer.


Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/uso terapêutico , Antígeno Ca-125/imunologia , Antígeno Ca-125/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/imunologia , Linfócitos T/metabolismo , Animais , Antígenos CD13/imunologia , Antígenos CD13/metabolismo , Feminino , Citometria de Fluxo , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Células Jurkat , Macaca fascicularis , Camundongos , Neoplasias Ovarianas/metabolismo , Linfócitos T/imunologia
7.
Sci Rep ; 5: 17943, 2015 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-26659273

RESUMO

Bispecific antibodies, while showing great therapeutic potential, pose formidable challenges with respect to their assembly, stability, immunogenicity, and pharmacodynamics. Here we describe a novel class of bispecific antibodies with native human immunoglobulin format. The design exploits differences in the affinities of the immunoglobulin isotypes for Protein A, allowing efficient large-scale purification. Using this format, we generated a bispecific antibody, REGN1979, targeting the B cell marker, CD20, and the CD3 component of the T cell receptor, which triggers redirected killing of B cells. In mice, this antibody prevented growth of B cell tumors and also caused regression of large established tumors. In cynomolgus monkeys, low doses of REGN1979 caused prolonged depletion of B cells in peripheral blood with a serum half-life of approximately 14 days. Further, the antibody induced a deeper depletion of B cells in lymphoid organs than rituximab. This format has broad applicability for development of clinical bispecific antibodies.


Assuntos
Anticorpos Biespecíficos/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Linfócitos B/imunologia , Citotoxicidade Imunológica , Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/isolamento & purificação , Anticorpos Biespecíficos/farmacologia , Antígenos CD20/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Complexo CD3/imunologia , Linhagem Celular Tumoral , Cromatografia de Afinidade , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Imunofenotipagem , Macaca fascicularis , Camundongos , Camundongos Knockout , Camundongos SCID , Modelos Moleculares , Dados de Sequência Molecular , Neoplasias/terapia , Fenótipo , Conformação Proteica , Alinhamento de Sequência , Linfócitos T/metabolismo , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/imunologia
8.
Cancer Cell ; 20(4): 472-86, 2011 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-22014573

RESUMO

Extensive crosstalk among ErbB/HER receptors suggests that blocking signaling from more than one family member may be essential to effectively treat cancer and limit drug resistance. We generated a conventional IgG molecule MEHD7945A with dual HER3/EGFR specificity by phage display engineering and used structural and mutational studies to understand how a single antigen recognition surface binds two epitopes with high affinity. As a human IgG1, MEHD7945A exhibited dual action by inhibiting EGFR- and HER3-mediated signaling in vitro and in vivo and the ability to engage immune effector functions. Compared with monospecific anti-HER antibodies, MEHD7945A was more broadly efficacious in multiple tumor models, showing that combined inhibition of EGFR and HER3 with a single antibody is beneficial.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos/uso terapêutico , Receptores ErbB/antagonistas & inibidores , Imunoglobulina G/uso terapêutico , Receptor ErbB-3/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/toxicidade , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Especificidade de Anticorpos , Antineoplásicos/química , Antineoplásicos/toxicidade , Sítios de Ligação de Anticorpos , Ligação Competitiva , Cetuximab , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/química , Receptores ErbB/imunologia , Feminino , Humanos , Imunoglobulina G/efeitos adversos , Imunoglobulina G/química , Sistema de Sinalização das MAP Quinases , Macaca fascicularis , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-3/química , Receptor ErbB-3/imunologia , Transdução de Sinais
9.
PLoS One ; 6(4): e17887, 2011 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-21526167

RESUMO

The antigen-binding site of Herceptin, an anti-human Epidermal Growth Factor Receptor 2 (HER2) antibody, was engineered to add a second specificity toward Vascular Endothelial Growth Factor (VEGF) to create a high affinity two-in-one antibody bH1. Crystal structures of bH1 in complex with either antigen showed that, in comparison to Herceptin, this antibody exhibited greater conformational variability, also called "structural plasticity". Here, we analyzed the biophysical and thermodynamic properties of the dual specific variants of Herceptin to understand how a single antibody binds two unrelated protein antigens. We showed that while bH1 and the affinity-improved bH1-44, in particular, maintained many properties of Herceptin including binding affinity, kinetics and the use of residues for antigen recognition, they differed in the binding thermodynamics. The interactions of bH1 and its variants with both antigens were characterized by large favorable entropy changes whereas the Herceptin/HER2 interaction involved a large favorable enthalpy change. By dissecting the total entropy change and the energy barrier for dual interaction, we determined that the significant structural plasticity of the bH1 antibodies demanded by the dual specificity did not translate into the expected increase of entropic penalty relative to Herceptin. Clearly, dual antigen recognition of the Herceptin variants involves divergent antibody conformations of nearly equivalent energetic states. Hence, increasing the structural plasticity of an antigen-binding site without increasing the entropic cost may play a role for antibodies to evolve multi-specificity. Our report represents the first comprehensive biophysical analysis of a high affinity dual specific antibody binding two unrelated protein antigens, furthering our understanding of the thermodynamics that drive the vast antigen recognition capacity of the antibody repertoire.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Antígenos/imunologia , Entropia , Proteínas Mutantes/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais Humanizados , Afinidade de Anticorpos/imunologia , Regiões Determinantes de Complementaridade/imunologia , Sequência Conservada , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Receptor ErbB-2/imunologia , Alinhamento de Sequência , Temperatura , Trastuzumab , Fator A de Crescimento do Endotélio Vascular/imunologia
10.
Methods Mol Biol ; 525: 353-76, xiii, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19252851

RESUMO

Affinity maturation is an important part of the therapeutic antibody development process as in vivo activity often requires high binding affinity. Here, we describe a targeted approach for affinity improvement of therapeutic antibodies. Sets of CDR residues that are solvent accessible and relatively diverse in natural antibodies are targeted for diversification. Degenerate oligonucleotides are used to generate combinatorial phage-displayed antibody libraries with varying degree of diversity at randomized positions from which high-affinity antibodies can be selected. An advantage of using antibodies for therapy is their exquisite target specificity, which enables selective antigen binding and reduces off-target effects. However, it can be useful, and often it is necessary, to generate cross-reactive antibodies binding to not only the human antigen but also the corresponding non-human primate or rodent orthologs. Such cross-reactive antibodies can be used to validate the therapeutic targeting and examine the safety profile in preclinical animal models before committing to a costly development track. We show how affinity improvement and cross-species binding can be achieved in a one-step process.


Assuntos
Anticorpos/uso terapêutico , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Biologia Molecular/métodos , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/genética , Sequência de Bases , Células Clonais , Códon/genética , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Fragmentos Fab das Imunoglobulinas/biossíntese , Imunoglobulina G/biossíntese , Concentração Inibidora 50 , Camundongos , Dados de Sequência Molecular , Oligonucleotídeos/genética , Biblioteca de Peptídeos
11.
Proc Natl Acad Sci U S A ; 102(24): 8466-71, 2005 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-15939870

RESUMO

Look-through mutagenesis (LTM) is a multidimensional mutagenesis method that simultaneously assesses and optimizes combinatorial mutations of selected amino acids. The process focuses on a precise distribution within one or more complementarity determining region (CDR) domains and explores the synergistic contribution of amino acid side-chain chemistry. LTM was applied to an anti-TNF-alpha antibody, D2E7, which is a challenging test case, because D2E7 was highly optimized (K(d) = 1 nM) by others. We selected and incorporated nine amino acids, representative of the major chemical functionalities, individually at every position in each CDR and across all six CDRs (57 aa). Synthetic oligonucleotides, each introducing one amino acid mutation throughout the six CDRs, were pooled to generate segregated libraries containing single mutations in one, two, and/or three CDRs for each V(H) and V(L) domain. Corresponding antibody libraries were displayed on the cell surface of yeast. After positive binding selection, 38 substitutions in 21 CDR positions were identified that resulted in higher affinity binding to TNF-alpha. These beneficial mutations in both V(H) and V(L) were represented in two combinatorial beneficial mutagenesis libraries and selected by FACS to produce a convergence of variants that exhibit between 500- and 870-fold higher affinities. Importantly, these enhanced affinities translate to a 15- to 30-fold improvement in in vitro TNF-alpha neutralization in an L929 bioassay. Thus, this LTM/combinatorial beneficial mutagenesis strategy generates a comprehensive energetic map of the antibody-binding site in a facile and rapid manner and should be broadly applicable to the affinity maturation of antibodies and other proteins.


Assuntos
Aminoácidos/química , Anticorpos/metabolismo , Afinidade de Anticorpos/genética , Sítios de Ligação de Anticorpos/genética , Biblioteca Gênica , Mutagênese Sítio-Dirigida/genética , Anticorpos/genética , Citometria de Fluxo , Mutação/genética , Testes de Neutralização , Fator de Necrose Tumoral alfa/metabolismo , Leveduras
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