RESUMEN
NKG2D is an activating receptor expressed by natural killer (NK) cells and other cytotoxic lymphocytes that plays a pivotal role in the elimination of neoplastic cells through recognition of different stress-induced cell surface ligands (NKG2DL). To employ this mechanism for cancer immunotherapy, we generated NKG2D-engaging bispecific antibodies that selectively redirect immune effector cells to cancer cells expressing the tumor-associated antigen ErbB2 (HER2). NKG2D-specific single chain fragment variable (scFv) antibodies cross-reactive toward the human and murine receptors were derived by consecutive immunization of chicken with the human and murine antigens, followed by stringent screening of a yeast surface display immune library. Four distinct species cross-reactive (sc) scFv domains were selected, and reformatted into a bispecific engager format by linking them via an IgG4 Fc domain to a second scFv fragment specific for ErbB2. The resulting molecules (termed scNKAB-ErbB2) were expressed as disulfide-linked homodimers, and demonstrated efficient binding to ErbB2-positive cancer cells as well as NKG2D-expressing primary human and murine lymphocytes, and NK-92 cells engineered with chimeric antigen receptors derived from human and murine NKG2D (termed hNKAR and mNKAR). Two of the scNKAB-ErbB2 molecules were found to compete with the natural NKG2D ligand MICA, while the other two engagers interacted with an epitope outside of the ligand binding site. Nevertheless, all four tested scNKAB-ErbB2 antibodies were similarly effective in redirecting the cytotoxic activity of primary human and murine lymphocytes as well as hNKAR-NK-92 and mNKAR-NK-92 cells to ErbB2-expressing targets, suggesting that further development of these species cross-reactive engager molecules for cancer immunotherapy is warranted.
Asunto(s)
Anticuerpos Biespecíficos , Reacciones Cruzadas , Células Asesinas Naturales , Subfamilia K de Receptores Similares a Lectina de Células NK , Receptor ErbB-2 , Animales , Humanos , Receptor ErbB-2/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Ratones , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Reacciones Cruzadas/inmunología , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/genética , Línea Celular Tumoral , Neoplasias/inmunología , Neoplasias/terapia , Inmunoterapia/métodosRESUMEN
Bispecific antibodies (BsAbs) have emerged as a major class of antibody therapeutics owing to their substantial potential in disease treatment. While several BsAbs have been successfully approved in recent years, ongoing development efforts continue to focus on optimizing various BsAbs tailored to particular antigens and action mechanisms, aiming to achieve favorable physicochemical properties. BsAbs generally encounter challenges due to their unfavorable physicochemical characteristics and poor manufacturing efficiencies, highlighting the need for optimization to achieve reliable productivity and developability. Herein, we describe the development of a novel symmetric BsAb, REGULGENT™ (N-term/C-term), comprising two Fab domains, using a common light chain. The heavy chain fragment encoded two antigen-binding determinants in one chain. The design and production of REGULGENT™ (N-term/C-term) are simple owing to the use of the same light chain, which does not induce heavy and light chain mispairing, frequently observed with the asymmetric BsAb format. REGULGENT™ (N-term/C-term) exhibited high expression and low aggregation characteristics during cell culture and stress treatment under low pH conditions. Differential scanning calorimetric data indicated that REGULGENT™ molecules had high conformational stability, similar to that of stabilized monoclonal antibodies. Surface plasmon resonance data showed that REGULGENT™ (N-term/C-term) could bind to two antigens simultaneously and exhibited a high affinity for two antigens. In summary, the symmetric BsAb format of REGULGENT™ confers its desirable IgG-like physicochemical properties, thus making it an excellent candidate for commercial development. The findings demonstrate a novel BsAb with substantial development potential for clinical applications.
Asunto(s)
Anticuerpos Biespecíficos , Ingeniería de Proteínas , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/genética , Humanos , Ingeniería de Proteínas/métodos , Estabilidad Proteica , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/metabolismo , AnimalesRESUMEN
Inherited bleeding disorders such as Glanzmann thrombasthenia (GT) lack prophylactic treatment options. As a result, serious bleeding episodes are treated acutely with blood product transfusions or frequent, repeated intravenous administration of recombinant activated coagulation factor VII (rFVIIa). Here we describe HMB-001, a bispecific antibody designed to bind and accumulate endogenous FVIIa and deliver it to sites of vascular injury by targeting it to the TREM (triggering receptor expressed on myeloid cells)-like transcript-1 (TLT-1) receptor that is selectively expressed on activated platelets. In healthy nonhuman primates, HMB-001 prolonged the half-life of endogenous FVIIa, resulting in its accumulation. Mouse bleeding studies confirmed antibody-mediated potentiation of FVIIa hemostatic activity by TLT-1 targeting. In ex vivo models of GT, HMB-001 localized FVIIa on activated platelets and potentiated fibrin-dependent platelet aggregation. Taken together, these results indicate that HMB-001 has the potential to offer subcutaneous prophylactic treatment to prevent bleeds in people with GT and other inherited bleeding disorders, with a low-frequency dosing regimen.
Asunto(s)
Anticuerpos Biespecíficos , Animales , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Biespecíficos/inmunología , Humanos , Factor VIIa , Plaquetas/metabolismo , Plaquetas/efectos de los fármacos , Plaquetas/inmunología , Ratones , Modelos Animales de Enfermedad , Hemorragia/prevención & control , Hemorragia/tratamiento farmacológico , Agregación Plaquetaria/efectos de los fármacos , Trombastenia/tratamiento farmacológico , Trombastenia/inmunología , Ratones Endogámicos C57BL , Femenino , Masculino , Macaca fascicularis , Activación Plaquetaria/efectos de los fármacosRESUMEN
Therapeutics for eradicating hepatitis B virus (HBV) infection are still limited and current nucleos(t)ide analogs (NAs) and interferon are effective in controlling viral replication and improving liver health, but they cannot completely eradicate the hepatitis B virus and only a very small number of patients are cured of it. The TCR-like antibodies recognizing viral peptides presented on human leukocyte antigens (HLA) provide possible tools for targeting and eliminating HBV-infected hepatocytes. Here, we generated three TCR-like antibodies targeting three different HLA-A2.1-presented peptides derived from HBV core and surface proteins. Bispecific antibodies (BsAbs) were developed by fuzing variable fragments of these TCR-like mAbs with an anti-CD3ϵ antibody. Our data demonstrate that the BsAbs could act as T cell engagers, effectively redirecting and activating T cells to target HBV-infected hepatocytes in vitro and in vivo. In HBV-persistent mice expressing human HLA-A2.1, two infusions of BsAbs induced marked and sustained suppression in serum HBsAg levels and also reduced the numbers of HBV-positive hepatocytes. These findings highlighted the therapeutic potential of TCR-like BsAbs as a new strategy to cure hepatitis B.
Asunto(s)
Anticuerpos Biespecíficos , Modelos Animales de Enfermedad , Virus de la Hepatitis B , Hepatitis B , Hepatocitos , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Hepatocitos/virología , Hepatocitos/inmunología , Ratones , Humanos , Virus de la Hepatitis B/inmunología , Virus de la Hepatitis B/genética , Hepatitis B/inmunología , Hepatitis B/virología , Antígeno HLA-A2/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunologíaRESUMEN
Recent developments in cancer immunotherapy have highlighted the potential of harnessing natural killer (NK) cells in the treatment of neoplastic malignancies. Of these, bispecific antibodies, and NK cell engager (NKCE) protein therapeutics in particular, have been of interest. Here, we used phage display and yeast surface display to engineer RLN131, a unique cross-reactive antibody that binds to human, mouse, and cynomolgus NKp46, an activating receptor found on NK cells. RLN131 induced proliferation and activation of primary NK cells, and was used to create bispecific NKCE constructs of varying configurations and valency. All NKCEs were able to promote greater NK cell cytotoxicity against tumor cells than an unmodified anti-CD20 monoclonal antibody, and activity was observed irrespective of whether the constructs contained a functional Fc domain. Competition binding and fine epitope mapping studies were used to demonstrate that RLN131 binds to a conserved epitope on NKp46, underlying its species cross-reactivity.
Asunto(s)
Células Asesinas Naturales , Receptor 1 Gatillante de la Citotoxidad Natural , Ingeniería de Proteínas , Células Asesinas Naturales/inmunología , Humanos , Receptor 1 Gatillante de la Citotoxidad Natural/genética , Receptor 1 Gatillante de la Citotoxidad Natural/inmunología , Animales , Ingeniería de Proteínas/métodos , Ratones , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/química , Reacciones CruzadasRESUMEN
Anti-cluster of differentiation (CD) 3 × α programmed death-ligand 1 (PD-L1) bispecific T-cell engager (BsTE)-bound T-cells (BsTE:T) are a promising new cancer treatment agent. However, the mechanisms of action of bispecific antibody-armed activated T-cells are poorly understood. Therefore, this study aimed to investigate the anti-tumor mechanism and efficacy of BsTE:T. The BsTE:T migration was assessed in vivo and in vitro using syngeneic and xenogeneic tumor models, flow cytometry, immunofluorescence staining, transwell migration assays, microfluidic chips, Exo View R100, western blotting, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology. In murine B16 melanoma, MC38 colon cancer, and human multiple myeloma cells, BsTE:T exhibited superior tumor elimination relative to that of T-cells or BsTE alone. Moreover, BsTE:T migration into tumors was significantly enhanced owing to the presence of PD-L1 in tumor cells and secretion of PD-L1-containing exosomes. Furthermore, increased infiltration of CD44highCD62Llow effector memory CD8+ T-cells into tumors was closely associated with the anti-tumor effect of BsTE:T. Therefore, BsTE:T is an innovative potential anti-tumor therapy, and exosomal PD-L1 plays a crucial role both in vitro and in vivo in the anti-tumor activity of BsTE:T.
Asunto(s)
Anticuerpos Biespecíficos , Antígeno B7-H1 , Exosomas , Animales , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/inmunología , Exosomas/metabolismo , Exosomas/inmunología , Ratones , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inhibidores , Humanos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ratones Endogámicos C57BL , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Complejo CD3/inmunología , Complejo CD3/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Línea Celular Tumoral , Femenino , Movimiento Celular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Anti-CTLA-4 antibodies faced challenges due to frequent adverse events and limited efficacy, which spurred the exploration of next-generation CTLA-4 therapeutics to balance regulatory T cells (Tregs) depletion and CD8 T cells activation. CCR8, identified primarily on tumor-infiltrating Tregs, has become a target of interest due to the anti-tumor effects demonstrated by CCR8 antibody-mediated Tregs depletion. Single-cell RNA sequencing analysis reveals that CCR8-positive Tregs constitute a small subset, with concurrent expression of CCR8 and CTLA-4. Consequently, we proposed a novel bispecific antibody targeting CCR8 and CTLA-4 that had the potential to enhance T cell activation while selectively depleting intratumor Tregs. The candidate molecule 2MW4691 was developed in a tetravalent symmetric format, maintaining a strong binding affinity for CCR8 while exhibiting relatively weaker CTLA-4 binding. This selective binding ability allowed 2MW4691 to target and deplete tumor-infiltrating Tregs with higher specificity. In vitro assays verified the antibody's capacity for antibody-dependent cellular cytotoxicity (ADCC) to Tregs with high level of CTLA-4 expression, but not CD8 T cells with relatively low level of CTLA-4 on cell surface. Also, 2MW4691 inhibited the CTLA-4 pathway and enhanced T cell activation. The in vivo therapeutic efficacy of 2MW4691 was further demonstrated using hCCR8 or hCTLA-4 humanized mouse models and hCCR8/hCTLA-4 double knock-in mouse models. In cynomolgus monkeys, 2MW4691 was well-tolerated, exhibited the anticipated pharmacokinetic profile, and had a minimal impact on the peripheral T cell population. The promising preclinical results supported the further evaluation of 2MW4691 as a next-generation Treg-based therapeutics in clinical trials.
Asunto(s)
Anticuerpos Biespecíficos , Linfocitos T CD8-positivos , Antígeno CTLA-4 , Linfocitos T Reguladores , Animales , Ratones , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/uso terapéutico , Humanos , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T CD8-positivos/inmunología , Receptores CCR8/inmunología , Neoplasias/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/terapia , Transducción de Señal/efectos de los fármacos , Femenino , Ensayos Antitumor por Modelo de Xenoinjerto , Macaca fascicularisRESUMEN
Multiple myeloma (MM) is a complex hematological malignancy characterized by abnormal antibody production from plasma cells. Despite advances in the treatment, many patients experience disease relapse or become refractory to treatment. G-protein-coupled receptor class C group 5 member D (GPRC5D), an orphan GPCR predominantly expressed in MM cells, is emerging as a promising target for MM immunotherapy. Talquetamab, a Food and Drug Administration-approved T-cell-directing bispecific antibody developed for treatment of MM, targets GPRC5D. Here, we elucidate the structure of GPRC5D complexed with the Fab fragment of talquetamab, using cryo-electron microscopy, providing the basis for recognition of GPRC5D by the bispecific antibody. GPRC5D forms a symmetric homodimer with the interface between transmembrane helix (TM) 4 of one protomer and TM4/5 of the other protomer. A single talquetamab Fab interacts with the GPRC5D dimer with its orientation toward the dimer interface. All six complementarity-determining regions of talquetamab engage with extracellular loops and TM3/5/7. In particular, the side-chain of an arginine residue from the antibody penetrates into a shallow pocket on the extracellular surface of GPRC5D. The structure offers insights for optimizing antibody design against GPRC5D for relapsed or refractory MM therapy.
Asunto(s)
Anticuerpos Biespecíficos , Microscopía por Crioelectrón , Mieloma Múltiple , Receptores Acoplados a Proteínas G , Mieloma Múltiple/inmunología , Humanos , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/metabolismo , Receptores Acoplados a Proteínas G/inmunología , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de ProteínaRESUMEN
Bispecific antibodies (bsAb) and multispecific antibodies (msAb) encompass a diverse variety of formats that can concurrently bind multiple epitopes, unlocking mechanisms to address previously difficult-to-treat or incurable diseases. Early assessment of candidate developability enables demotion of antibodies with low potential and promotion of the most promising candidates for further development. Protein-based therapies have a stringent set of developability requirements in order to be competitive (e.g. high-concentration formulation, and long half-life) and their assessment requires a robust toolkit of methods, few of which are validated for interrogating bsAbs/msAbs. Important considerations when assessing the developability of bsAbs/msAbs include their molecular format, likelihood for immunogenicity, specificity, stability, and potential for high-volume production. Here, we summarize the critical aspects of developability assessment, and provide guidance on how to develop a comprehensive plan tailored to a given bsAb/msAb.
Asunto(s)
Anticuerpos Biespecíficos , Anticuerpos Biespecíficos/inmunología , Humanos , AnimalesRESUMEN
Current medical therapies for treating acute myeloid leukemia (AML) remain unmet, and AML patients may benefit from targeted immunotherapy approaches that focus on specific tumor antigens. GRP78, which is upregulated in various malignant tumors such as AML, is partially expressed as cell surface GRP78 (csGRP78) on the cell membrane, making it an ideal target for redirecting T cells, including T-cell engagers. However, considering the conventional approach of using two scFv segments to construct a bispecific T-cell engager (BiTE), we have undertaken the development of a novel BiTE that utilizes a cyclic peptide ligand to specifically target csGRP78, which we refer to as GRP78-CD3/BiTE. We studied the effects of GRP78-CD3/BiTE on treatments for AML in vitro and in vivo and assessed the pharmacokinetics of this engager. Our findings demonstrated that GRP78-CD3/BiTE could not only effectively mediate the cytotoxicity of T cells against csGRP78-expressing AML cells but also specifically eliminate primary AML tumor cells in vitro. Furthermore, GRP78-CD3/BiTE exhibited a longer half-life despite having a lower molecular weight than CD19-CD3/BiTE. In a xenograft mouse model of AML, treatment with GRP78-CD3/BiTE prolonged the survival time of the mice. Our findings demonstrate that GRP78-CD3/BiTE is effective and selective for eliminating csGRP78-expressing AML cells and suggest that this approach to targeted immunotherapy could lead to effective new treatments for AML.
Asunto(s)
Anticuerpos Biespecíficos , Chaperón BiP del Retículo Endoplásmico , Leucemia Mieloide Aguda , Linfocitos T , Humanos , Animales , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/tratamiento farmacológico , Ratones , Linfocitos T/inmunología , Linfocitos T/metabolismo , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Complejo CD3/inmunología , Proteínas de Choque Térmico/inmunología , Proteínas de Choque Térmico/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Línea Celular Tumoral , Ligandos , Femenino , Ratones SCID , Inmunoterapia/métodos , Ratones Endogámicos NODRESUMEN
T cell engagers (TCEs) are becoming an integral class of biological therapeutic owing to their highly potent ability to eradicate cancer cells. Nevertheless, the widespread utility of classical CD3-targeted TCEs has been limited by narrow therapeutic index (TI) linked to systemic CD4+ T cell activation and aberrant cytokine release. One attractive approach to circumvent the systemic activation of pan CD3+ T cells and reduce the risk of cytokine release syndrome is to redirect specific subsets of T cells. A promising strategy is the use of peptide-major histocompatibility class I bispecific antibodies (pMHC-IgGs), which have emerged as an intriguing modality of TCE, based on their ability to selectively redirect highly reactive viral-specific effector memory cytotoxic CD8+ T cells to eliminate cancer cells. However, the relatively low frequency of these effector memory cells in human peripheral blood mononuclear cells (PBMCs) may hamper their redirection as effector cells for clinical applications. To mitigate this potential limitation, we report here the generation of a pMHC-IgG derivative known as guided-pMHC-staging (GPS) carrying a covalent fusion of a monovalent interleukin-2 (IL-2) mutein (H16A, F42A). Using an anti-epidermal growth factor receptor (EGFR) arm as a proof-of-concept, tumor-associated antigen paired with a single-chain HLA-A *02:01/CMVpp65 pMHC fusion moiety, we demonstrate in vitro that the IL-2-armored GPS modality robustly expands CMVpp65-specific CD8+ effector memory T cells and induces potent cytotoxic activity against target cancer cells. Similar to GPS, IL-2-armored GPS molecules induce modulated T cell activation and reduced cytokine release profile compared to an analogous CD3-targeted TCE. In vivo we show that IL-2-armored GPS, but not the corresponding GPS, effectively expands grafted CMVpp65 CD8+ T cells from unstimulated human PBMCs in an NSG mouse model. Lastly, we demonstrate that the IL-2-armored GPS modality exhibits a favorable developability profile and monoclonal antibody-like pharmacokinetic properties in human neonatal Fc receptor transgenic mice. Overall, IL-2-armored GPS represents an attractive approach for treating cancer with the potential for inducing vaccine-like antiviral T cell expansion, immune cell redirection as a TCE, and significantly widened TI due to reduced cytokine release.
Asunto(s)
Anticuerpos Biespecíficos , Linfocitos T CD8-positivos , Interleucina-2 , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Humanos , Animales , Ratones , Linfocitos T CD8-positivos/inmunología , Interleucina-2/inmunología , Citocinas/inmunología , Citocinas/metabolismo , Proteínas de la Matriz Viral/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Línea Celular Tumoral , Memoria Inmunológica , Antígenos de Histocompatibilidad Clase I/inmunologíaRESUMEN
Monoclonal neutralizing antibodies (mAbs) are considered an important prophylactic against SARS-CoV-2 infection in at-risk populations and a strategy to counteract future sarbecovirus-induced disease. However, most mAbs isolated so far neutralize only a few sarbecovirus strains. Therefore, there is a growing interest in bispecific antibodies (bsAbs) which can simultaneously target different spike epitopes and thereby increase neutralizing breadth and prevent viral escape. Here, we generate and characterize a panel of 30 novel broadly reactive bsAbs using an efficient controlled Fab-arm exchange protocol. We specifically combine some of the broadest mAbs described so far, which target conserved epitopes on the receptor binding domain (RBD). Several bsAbs show superior cross-binding and neutralization compared to the parental mAbs and cocktails against sarbecoviruses from diverse clades, including recent SARS-CoV-2 variants. BsAbs which include mAb COVA2-02 are among the most potent and broad combinations. As a result, we study the unknown epitope of COVA2-02 and show that this mAb targets a distinct conserved region at the base of the RBD, which could be of interest when designing next-generation bsAb constructs to contribute to a better pandemic preparedness.
Asunto(s)
Anticuerpos Biespecíficos , Anticuerpos Neutralizantes , Anticuerpos Antivirales , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Anticuerpos Biespecíficos/inmunología , Humanos , SARS-CoV-2/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Neutralizantes/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , COVID-19/inmunología , COVID-19/prevención & control , Epítopos/inmunología , Pruebas de Neutralización , Animales , Anticuerpos Monoclonales/inmunologíaRESUMEN
The mechanism of action of bispecific antibodies (bsAbs) directing T-cell immunity to solid tumors is incompletely understood. Here, we screened a series of CD3xHER2 bsAbs using extracellular matrix (ECM) embedded breast cancer tumoroid arrays exposed to healthy donor-derived T-cells. An initial phase of random T-cell movement throughout the ECM (day 1-2), was followed by a bsAb-dependent phase of active T-cell recruitment to tumoroids (day 2-4), and tumoroid killing (day 4-6). Low affinity HER2 or CD3 arms were compensated for by increasing bsAb concentrations. Instead, a bsAb binding a membrane proximal HER2 epitope supported tumor killing whereas a bsAb binding a membrane distal epitope did not, despite similar affinities and intra-tumoroid localization of the bsAbs, and efficacy in 2D co-cultures. Initial T-cell-tumor contact through effective bsAbs triggered a wave of subsequent T-cell recruitment. This critical surge of T-cell recruitment was explained by paracrine signaling and preceded a full-scale T-cell tumor attack.
Asunto(s)
Anticuerpos Biespecíficos , Complejo CD3 , Comunicación Paracrina , Linfocitos T , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/inmunología , Humanos , Complejo CD3/inmunología , Complejo CD3/metabolismo , Linfocitos T/inmunología , Femenino , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Línea Celular TumoralRESUMEN
The growing clinical success of bispecific antibodies (bsAbs) has led to rapid interest in leveraging dual targeting in order to generate novel modes of therapeutic action beyond mono-targeting approaches. While bsAbs that bind targets on two different cells (trans-targeting) are showing promise in the clinic, the co-targeting of two proteins on the same cell surface through cis-targeting bsAbs (cis-bsAbs) is an emerging strategy to elicit new functionalities. This includes the ability to induce proximity, enhance binding to a target, increase target/cell selectivity, and/or co-modulate function on the cell surface with the goal of altering, reversing, or eradicating abnormal cellular activity that contributes to disease. In this review, we focus on the impact of cis-bsAbs in the clinic, their emerging applications, and untangle the intricacies of improving bsAb discovery and development.
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Anticuerpos Biespecíficos , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/inmunología , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , AnimalesRESUMEN
The precise regulation of protein function is essential in biological systems and a key goal in chemical biology and protein engineering. Here, we describe a straightforward method to engineer functional control into the isopeptide bond-forming SpyTag/SpyCatcher protein ligation system. First, we perform a cysteine scan of the structured region of SpyCatcher. Except for two known reactive and catalytic residues, none of these mutations abolish reactivity. In a second screening step, we modify the cysteines with disulfide bond-forming small molecules. Here we identify 8 positions at which modifications strongly inhibit reactivity. This inhibition can be reversed by reducing agents. We call such a reversibly inhibitable SpyCatcher "SpyLock". Using "BiLockCatcher", a genetic fusion of wild-type SpyCatcher and SpyLock, and SpyTagged antibody fragments, we generate bispecific antibodies in a single, scalable format, facilitating the screening of a large number of antibody combinations. We demonstrate this approach by screening anti-PD-1/anti-PD-L1 bispecific antibodies using a cellular reporter assay.
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Anticuerpos Biespecíficos , Cisteína , Ingeniería de Proteínas , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/química , Humanos , Ingeniería de Proteínas/métodos , Cisteína/química , Cisteína/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Células HEK293 , Disulfuros/química , AnimalesRESUMEN
Off-the-shelf T-cell-redirecting bispecific antibodies targeting BCMA, GPRC5D, and FcRH5 have high activity in multiple myeloma with a manageable toxicity profile. However, not all patients respond to bispecific antibodies and patients can develop bispecific antibody resistance after an initial response. Mechanisms that contribute to bispecific antibody resistance are multifactorial and include tumour-related factors, such as high tumour burden, expression of T-cell inhibitory ligands, and antigen loss. Resistance due to antigen escape can be prevented by simultaneously targeting two tumour-associated antigens with a trispecific antibody or a combination of two bispecific antibodies. There is also increasing evidence that primary resistance to bispecific antibodies is associated with impaired baseline T-cell function. Long-term exposure to bispecific antibodies with chronic T-cell stimulation further aggravates T-cell dysfunction, which could contribute to failure of disease control. Therapeutic interference with T-cell exhaustion by targeting inhibitory or costimulatory pathways could improve bispecific antibody-mediated antitumour activity. The immunosuppressive microenvironment also contributes to bispecific antibody resistance. CD38-targeting antibodies hold promise as combination partners for bispecific antibodies because of their potential to eliminate CD38+ immune suppressor cells. In conclusion, a better understanding of the mechanisms underlying the absence of disease response has provided novel insights to optimise T-cell activity and bispecific antibody efficacy in multiple myeloma.
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Anticuerpos Biespecíficos , Mieloma Múltiple , Linfocitos T , Mieloma Múltiple/inmunología , Mieloma Múltiple/terapia , Mieloma Múltiple/tratamiento farmacológico , Humanos , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Linfocitos T/inmunología , Resistencia a AntineoplásicosRESUMEN
INTRODUCTION: Since the approval of the bispecific antibody blinatumomab in 2017 for the treatment of acute lymphoblastic leukemia in relapse, the development of numerous bispecific antibody constructs has dramatically expanded in hematologic malignancies. Many have recently received Food Drug Administration and European Medicines Agency approvals in various stages of treatment for lymphomas, leukemias, and multiple myeloma. AREAS COVERED: The purpose of this review is to provide an overview of bispecific antibody treatment including the mechanisms leading to effector T cells targeting tumor-associated antigens, the treatment indications, efficacies, toxicities, and challenges of the different constructs. A literature search was performed through access to PubMed and clinicaltrials.gov. EXPERT OPINION: While there has been substantial success in the treatment of NHL, MM, and ALL, there are still hematologic malignancies such as AML where there has been limited progress. It is important to continue to investigate new designs, tumor antigen targets, and further refine where current approved bispecific antibodies fit in terms of sequencing of therapy. Hopefully, with the knowledge gained in recent years and the explosion of these therapies, patients with blood cancers will continue to benefit from these treatments for years to come.
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Anticuerpos Biespecíficos , Complejo CD3 , Neoplasias Hematológicas , Linfocitos T , Humanos , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Biespecíficos/inmunología , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/tratamiento farmacológico , Linfocitos T/inmunología , Linfocitos T/efectos de los fármacos , Complejo CD3/inmunología , Complejo CD3/antagonistas & inhibidores , AnimalesRESUMEN
T-cell engaging (TCE) bispecific antibodies are potent drugs that trigger the immune system to eliminate cancer cells, but administration can be accompanied by toxic side effects that limit dosing. TCEs function by binding to cell surface receptors on T cells, frequently CD3, with one arm of the bispecific antibody while the other arm binds to cell surface antigens on cancer cells. On-target, off-tumor toxicity can arise when the target antigen is also present on healthy cells. The toxicity of TCEs may be ameliorated through the use of pro-drug forms of the TCE, which are not fully functional until recruited to the tumor microenvironment. This can be accomplished by masking the anti-CD3 arm of the TCE with an autoinhibitory motif that is released by tumor-enriched proteases. Here, we solve the crystal structure of the antigen-binding fragment of a novel anti-CD3 antibody, E10, in complex with its epitope from CD3 and use this information to engineer a masked form of the antibody that can activate by the tumor-enriched protease matrix metalloproteinase 2 (MMP-2). We demonstrate with binding experiments and in vitro T-cell activation and killing assays that our designed prodrug TCE is capable of tumor-selective T-cell activity that is dependent upon MMP-2. Furthermore, we demonstrate that a similar masking strategy can be used to create a pro-drug form of the frequently used anti-CD3 antibody SP34. This study showcases an approach to developing immune-modulating therapeutics that prioritizes safety and has the potential to advance cancer immunotherapy treatment strategies.
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Anticuerpos Biespecíficos , Complejo CD3 , Inmunoterapia , Profármacos , Linfocitos T , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Humanos , Complejo CD3/inmunología , Inmunoterapia/métodos , Linfocitos T/inmunología , Profármacos/farmacología , Profármacos/química , Neoplasias/inmunología , Neoplasias/terapia , Neoplasias/tratamiento farmacológico , Ingeniería de Proteínas/métodos , Metaloproteinasa 2 de la Matriz/inmunologíaRESUMEN
Background: CD38 and CD47 are expressed in many hematologic malignancies, including multiple myeloma (MM), B-cell non-Hodgkin lymphoma (NHL), B-cell acute lymphoblastic leukemia (ALL), and B-cell chronic lymphocytic leukemia (CLL). Here, we evaluated the antitumor activities of CD38/CD47 bispecific antibodies (BsAbs). Methods: Five suitable anti-CD38 antibodies for co-targeting CD47 and CD38 BsAb were developed using a 2 + 2 "mAb-trap" platform. The activity characteristics of the CD38/CD47 BsAbs were evaluated using in vitro and in vivo systems. Results: Using hybridoma screening technology, we obtained nine suitable anti-CD38 antibodies. All anti-CD38 antibodies bind to CD38+ tumor cells and kill tumor cells via antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Five anti-CD38 antibodies (4A8, 12C10, 26B4, 35G5, and 65A7) were selected for designing CD38/CD47 BsAbs (IMM5605) using a "mAb-trap" platform. BsAbs had higher affinity and binding activity to the CD38 target than those to the CD47 target, decreasing the potential on-target potential and off-tumor effects. The CD38/CD47 BsAbs did not bind to RBCs and did not induce RBC agglutination; thus, BsAbs had much lower blood toxicity. The CD38/CD47 BsAbs had a greater ability to block the CD47/SIRPα signal in CD38+/CD47+ tumor cells than IMM01 (SIRPα Fc fusion protein). Through Fc domain engineering, CD38/CD47 BsAbs were shown to kill tumors more effectively by inducing ADCC and ADCP. IMM5605-26B4 had the strongest inhibitory effect on cellular CD38 enzymatic activity. IMM5605-12C10 had the strongest ability to directly induce the apoptosis of tumor cells. The anti-CD38 antibody 26B4 combined with the SIRPα-Fc fusion proteins showed strong antitumor effects, which were better than any of the mono-therapeutic agents used alone in the NCI-H929 cell xenograft model. The CD38/CD47 BsAbs exhibited strong antitumor effects; specifically, IMM5605-12C10 efficiently eradicated all established tumors in all mice. Conclusion: A panel of BsAbs targeting CD38 and CD47 developed based on the "mAb-tarp" platform showed potent tumor-killing ability in vitro and in vivo. As BsAbs had lower affinity for binding to CD47, higher affinity for binding to CD38, no affinity for binding to RBCs, and did not induce RBC agglutination, we concluded that CD38/CD47 BsAbs are safe and have a satisfactory tolerability profile.
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ADP-Ribosil Ciclasa 1 , Antígeno CD47 , Neoplasias Hematológicas , Antígeno CD47/inmunología , Antígeno CD47/antagonistas & inhibidores , Antígeno CD47/metabolismo , ADP-Ribosil Ciclasa 1/antagonistas & inhibidores , ADP-Ribosil Ciclasa 1/inmunología , ADP-Ribosil Ciclasa 1/metabolismo , Humanos , Animales , Ratones , Neoplasias Hematológicas/terapia , Neoplasias Hematológicas/inmunología , Línea Celular Tumoral , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/antagonistas & inhibidores , Citotoxicidad Celular Dependiente de Anticuerpos , Femenino , Antineoplásicos Inmunológicos/farmacologíaRESUMEN
SARS-CoV-2 has been evolving into a large number of variants, including the highly pathogenic Delta variant, and the currently prevalent Omicron subvariants with extensive evasion capability, which raises an urgent need to develop new broad-spectrum neutralizing antibodies. Herein, we engineer two IgG-(scFv)2 form bispecific antibodies with overlapping epitopes (bsAb1) or non-overlapping epitopes (bsAb2). Both bsAbs are significantly superior to the parental monoclonal antibodies in terms of their antigen-binding and virus-neutralizing activities against all tested circulating SARS-CoV-2 variants including currently dominant JN.1. The bsAb1 can efficiently neutralize all variants insensitive to parental monoclonal antibodies or the cocktail with IC50 lower than 20â ng/mL, even slightly better than bsAb2. Furthermore, the cryo-EM structures of bsAb1 in complex with the Omicron spike protein revealed that bsAb1 with overlapping epitopes effectively locked the S protein, which accounts for its conserved neutralization against Omicron variants. The bispecific antibody strategy engineered from overlapping epitopes provides a novel solution for dealing with viral immune evasion.