Inhaled anti-TSLP antibody fragment, ecleralimab, blocks responses to allergen in mild asthma.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is a key upstream regulator driving allergic inflammatory responses. We evaluated the efficacy and safety of ecleralimab, a potent inhaled neutralising antibody fragment against human TSLP, using allergen inhalation challenge (AIC) in subjects with mild atopic asthma. METHODS: This was a 12-week, randomised, double-blind, placebo-controlled, parallel-design, multicentre allergen bronchoprovocation study conducted at 10 centres across Canada and Germany. Subjects aged 18-60 years with stable mild atopic asthma were randomised (1:1) to receive 4 mg once-daily inhaled ecleralimab or placebo. Primary end-points were the allergen-induced change in forced expiratory volume in 1 s (FEV1) during the late asthmatic response (LAR) measured by area under the curve (AUC3-7h) and maximum percentage decrease (LAR%) on day 84, and the safety of ecleralimab. Allergen-induced early asthmatic response (EAR), sputum eosinophils and fractional exhaled nitric oxide (F ENO) were secondary and exploratory end-points. RESULTS: 28 subjects were randomised to ecleralimab (n=15) or placebo (n=13). On day 84, ecleralimab significantly attenuated LAR AUC3-7h by 64% (p=0.008), LAR% by 48% (p=0.029), and allergen-induced sputum eosinophils by 64% at 7 h (p=0.011) and by 52% at 24 h (p=0.047) post-challenge. Ecleralimab also numerically reduced EAR AUC0-2h (p=0.097) and EAR% (p=0.105). F ENO levels were significantly reduced from baseline throughout the study (p<0.05), except at 24 h post-allergen (day 43 and day 85). Overall, ecleralimab was safe and well tolerated. CONCLUSION: Ecleralimab significantly attenuated allergen-induced bronchoconstriction and airway inflammation, and was safe in subjects with mild atopic asthma.

Development of an inhaled anti-TSLP therapy for asthma.

Thymic stromal lymphopoietin (TSLP), an epithelial cell-derived cytokine, acts as a key mediator in airway inflammation and modulates the function of multiple cell types, including dendritic cells and group 2 innate lymphoid cells. TSLP plays a role in asthma pathogenesis as an upstream cytokine, and data suggest that TSLP blockade with the anti-TSLP monoclonal antibody, tezepelumab, could be efficacious in a broad asthma population. Currently approved asthma biologic therapies target allergic or eosinophilic disease and require phenotyping; therefore, an unmet need exists for a therapy that can address Type 2 (T2)-high and T2-low inflammation in asthma. All currently approved biologic treatments are delivered intravenously or subcutaneously; an inhaled therapy route that allows direct targeting of the lung with reduced systemic impact may offer advantages. Currently in development, ecleralimab (CSJ117) represents the first inhaled anti-TSLP antibody fragment that binds soluble TSLP and prevents TSLP receptor activation, thereby inhibiting further inflammatory signalling cascades. This anti-TSLP antibody fragment is being developed for patients with severe uncontrolled asthma despite standard of care inhaled therapy. A Phase IIa proof of concept study, using allergen bronchoprovocation as a model for asthma exacerbations, found that ecleralimab was well-tolerated and reduced allergen-induced bronchoconstriction in adult patients with mild asthma. These results suggest ecleralimab may be a promising, new therapeutic class for asthma treatment.

The Effect of Microwave Ablation Combined with Anti-PD-1 Monoclonal Antibody on T Cell Subsets and Long-Term Prognosis in Patients Suffering from Non-Small-Cell Lung Cancer.

Objective: This research is aimed at studying the effect of microwave ablation combined with the antiprogrammed death- (PD-) 1 monoclonal antibody on T cell subsets and long-term prognosis in patients suffering from non-small-cell lung cancer (NSCLC). Methods: Employing the random number table technique, a total of 122 NSCLC patients who received treatment at our hospital between May 2015 and June 2019 were selected and assigned to the observation group and the control group, and each group comprised 61 patients (n = 61). While the control group received only anti-PD-1 monoclonal antibody treatment, the observation group received microwave ablation in combination with anti-PD-1 monoclonal antibody. The clinical efficacy was observed for both groups. The levels of T cell subsets (CD3+, CD4+, and CD8+), serum tumor markers (squamous cell carcinoma antigen (SCCA), cytokeratin Ig fragment (CYFRA21-1), and serum carcinoembryonic antigen (CEA)), nuclear factor kappa B (NF-κB), protease C (PKC), and mitogen-activated protein kinase (MAPK) mRNA expression between the two groups were compared. The frequency of adverse reactions was observed in both groups. The survival time of both the groups was recorded over the course of three years of follow-up. The Kaplan-Meier method was employed for analyzing the survival of both the control and the observation group. Results: The response rate (RR) of the observation group (80.33%) was considerably greater in comparison to that of the control group (62.30%) (P < 0.05). Following treatment, the observation group's levels of CD3+, CD4+, CD8+, SCCA, CyFRA21-1, and CEA and the mRNA expressions of NF-κB, PKC, and MAPK were superior to those of the control group, with statistical significances (all P < 0.05). Between the two groups, there was no significant difference in the occurrence of adverse reactions (P > 0.05). The observation group had greater 1-, 2-, and 3-year survival rates (57.38%, 39.34%, and 29.51%) than the control group (32.79%, 18.03%, and 8.20%), with statistically significant differences (all P < 0.05). Conclusion: Microwave ablation in combination with an anti-PD-1 monoclonal antibody could effectively improve the level of T cell subsets and serum tumor markers in NSCLC patients, resulting in a long-term prognosis of patients with good therapeutic effect and safety.

Evaluation of an 131I-labeled HER2-specific single domain antibody fragment for the radiopharmaceutical therapy of HER2-expressing cancers.

Radiopharmaceutical therapy (RPT) is an attractive strategy for treatment of disseminated cancers including those overexpressing the HER2 receptor including breast, ovarian and gastroesophageal carcinomas. Single-domain antibody fragments (sdAbs) exemplified by the HER2-targeted VHH_1028 evaluated herein are attractive for RPT because they rapidly accumulate in tumor and clear faster from normal tissues than intact antibodies. In this study, VHH_1028 was labeled using the residualizing prosthetic agent N-succinimidyl 3-guanidinomethyl 5-[131I]iodobenzoate (iso-[131I]SGMIB) and its tissue distribution evaluated in the HER2-expressing SKOV-3 ovarian and BT474 breast carcinoma xenograft models. In head-to-head comparisons to [131I]SGMIB-2Rs15d, a HER2-targeted radiopharmaceutical currently under clinical investigation, iso-[131I]SGMIB-VHH_1028 exhibited significantly higher tumor uptake and significantly lower kidney accumulation. The results demonstrated 2.9 and 6.3 times more favorable tumor-to-kidney radiation dose ratios in the SKOV-3 and BT474 xenograft models, respectively. Iso-[131I]SGMIB-VHH_1028 was prepared using a solid-phase extraction method for purification of the prosthetic agent intermediate Boc2-iso-[131I]SGMIB that reproducibly scaled to therapeutic-level doses and obviated the need for its HPLC purification. Single-dose (SKOV-3) and multiple-dose (BT474) treatment regimens demonstrated that iso-[131I]SGMIB-VHH_1028 was well tolerated and provided significant tumor growth delay and survival prolongation. This study suggests that iso-[131I]SGMIB-VHH_1028 is a promising candidate for RPT of HER2-expressing cancers and further development is warranted.

A Novel and Potent Thrombolytic Fusion Protein Consisting of Anti-Insoluble Fibrin Antibody and Mutated Urokinase.

Tissue plasminogen activator (tPA) is used clinically because it has a higher binding specificity for insoluble fibrin (IF) than urokinase (UK), but even pro-tPA has catalytic activity against substrates other than IF. UK has the advantage that it is specifically activated on IF; however, it binds IF weakly. Previously, we established a monoclonal antibody (mAb) that recognizes a pit structure formed only in IF. Here, we developed a new mAb against the pit, 1101, that does not affect coagulation or fibrinolysis, and prepared a fusion protein of UK with humanized 1101 Fab to transport UK selectively to IF. In IF-containing lesions, UK is cleaved by plasmin at two sites, Lys158/Ile159 and Lys135/Lys136. Cleavage of the former leads to activation of UK; however, because activated UK is linked by S-S bonds before and after cleavage, it is not released from the fusion. Cleavage at the latter site causes UK to leave the fusion protein; hence, we mutated Lys135/Lys136 to Gly135/Gly136 to prevent release of UK. This engineered UK-antibody fusion, AMU1114, significantly decreased the reduction of plasma plasminogen levels in vivo relative to UK. In a photochemically induced mouse model of thrombus, the vascular patency rate was 0% (0/10) in the control, 50% (5/10) in the tPA treatment group, and 90% (9/10) in the AMU1114 treatment group. Although no death was observed 1 hour after administration of each thrombolytic agent, some mice died within 24 hours in all treatment groups, including control. These data indicate the need for further basic studies of AMU1114.

Population pharmacokinetic-pharmacodynamic modeling of PB2452, a monoclonal antibody fragment being developed as a ticagrelor reversal agent, in healthy volunteers.

PB2452, a neutralizing monoclonal antibody fragment that binds the antiplatelet drug ticagrelor with high affinity, is being developed as a ticagrelor reversal agent. To identify a clinically useful intravenous (i.v.) reversal regimen, a semimechanistic exposure-response model was developed during the PB2452 first-in-human phase I study. From a randomized, double-blind, placebo-controlled, single-dose trial to evaluate the safety, efficacy, and pharmacokinetics (PKs) of PB2452 in 61 healthy volunteers pretreated with ticagrelor, sequential dose cohort data were used to build and refine an exposure-response model that combined population PK models for ticagrelor (TICA), ticagrelor active metabolite (TAM), and PB2452, and related their binding relationships to the PK of uncomplexed TICA and TAM which is predictive of platelet inhibition. Platelet function was assessed by multiple assays. The model was developed using Bayesian methods in NONMEM. Human PK and pharmacodynamic data from sequential dose cohorts were used to initially define and then refine model parameters. Model simulations indicated that an initial i.v. bolus of PB2452, followed by a high-rate infusion, and then a slower-rate infusion would provide immediate and sustained reversal of the antiplatelet effects of ticagrelor. Based on model predictions, a 6 g i.v. bolus followed by 6 g infused over 4 h and then 6 g over 12 h was identified and tested in study subjects and shown to provide complete reversal within 5 min of infusion onset that was sustained for 20-24 h. The model is predictive of the reversal profile of PB2452 and will inform future trials of PB2452.

Novel Dual-Targeting Antibody Fragment IDB0062 Overcomes Anti-Vascular Endothelial Growth Factor Drug Limitations in Age-Related Macular Degeneration.

Purpose: Repeated administration of anti-vascular endothelial growth factor drugs to treat age-related macular degeneration leads to resistance. To overcome this drawback, we developed the novel recombinant dual-targeting antibody fragment IDB0062, which is comprised of the anti-vascular endothelial growth factor A Fab and neuropilin 1-targeting peptide, and we assessed its properties. Methods: We compared the in vitro activity of IDB0062 and conventional drugs using cell proliferation, wound healing, and Transwell assays. The in vivo efficacy of IDB0062 was determined using mouse choroidal neovascularization and oxygen-induced retinopathy models. To evaluate the ocular distribution of IDB0062, we intravitreally administered IDB0062 and ranibizumab to cynomolgus monkeys and measured the retinal drug levels. Results: IDB0062 effectively inhibited not only vascular endothelial growth factor A in vitro but also placenta growth factor 2, vascular endothelial growth factor B, and platelet-derived growth factor BB, which induce vascular endothelial growth factor A-independent angiogenesis. In addition, IDB0062 showed non-inferior efficacy compared with aflibercept in vivo despite the low selectivity for mouse vascular endothelial growth factor A. In the monkey intravitreal pharmacokinetic study, IDB0062 improved drug distribution in the retina compared with ranibizumab, confirming the accelerated onset of pharmacological action when IDB0062 is injected in the vitreous humor. Conclusions: Through neuropilin 1 binding, IDB0062 can improve the efficacy and accelerate the onset of pharmacological action in the posterior segment, which is targeted for macular degeneration, thereby improving drug responsiveness in drug-resistant patients. Translational Relevance: Considering its novel mechanism of action, IDB0062 may help in controlling resistance to conventional anti-vascular endothelial growth factor drugs in clinical settings.

Human Antibody Domains and Fragments Targeting Neutrophil Elastase as Candidate Therapeutics for Cancer and Inflammation-Related Diseases.

Neutrophil elastase (NE) is a serine protease released during neutrophil maturation. High levels of NE are related to lung tissue damage and poor prognosis in cancer; thus, NE is a potential target for therapeutic immunotherapy for multiple lung diseases and cancers. Here, we isolate and characterize two high-affinity, specific, and noncompetitive anti-NE antibodies Fab 1C10 and VH 1D1.43 from two large phage-displayed human Fab and VH libraries. After fusion with human IgG1 Fc, both of them (VH-Fc 1D1.43 and IgG1 1C10) inhibit NE enzymatic activity with VH-Fc 1D1.43 showing comparable inhibitory effects to that of the small molecule NE inhibitor SPCK and IgG1 1C10 exhibiting even higher (2.6-fold) activity than SPCK. Their epitopes, as mapped by peptide arrays combined with structural modeling, indicate different mechanisms for blocking NE activity. Both VH-Fc and IgG1 antibodies block NE uptake by cancer cells and fibroblast differentiation. VH-Fc 1D1.43 and IgG1 1C10 are promising for the antibody-based immunotherapy of cancer and inflammatory diseases.

A Two-Pore Physiologically Based Pharmacokinetic Model to Predict Subcutaneously Administered Different-Size Antibody/Antibody Fragments.

Quantitative modeling of the subcutaneous absorption processes of protein therapeutics is challenging. Here we have proposed a "two-pore" PBPK model that is able to simultaneously characterize plasma PK of different-size protein therapeutics in mice. The skin compartment is evolved to mechanistically account for the absorption pathways through lymph and blood capillaries, as well as local degradation at the SC injection site. The model is developed using in-house plasma PK data generated following subcutaneous administration of 6 different-size protein therapeutics (13-150 kDa) in mice. The model was able to capture plasma PK of all molecules following intravenous and subcutaneous administration relatively well. From the observed plasma PK profiles, as well as from the model simulation result, several important PK descriptors were found to be dependent on protein size for FcRn nonbinding molecules. A positive correlation was found between Tmax and protein size. A "U" shape relationship was found between Cmax and protein size. Negative correlations were observed between bioavailability (F) and local degradation rate (kdeg,SC), and F and protein size. Pathway analysis of the model was conducted for the subcutaneous absorption process, and continuous relationships were established between the percentage of absorption through lymphatic and vascular pathways and protein size. This PBPK model could serve as a platform for the development of different-size protein therapeutics and will be scaled up to humans for translational studies in the future.

Brolucizumab: a novel anti-VEGF humanized single-chain antibody fragment for treating w-AMD.

INTRODUCTION: Wet age-related macular degeneration (w-AMD) represents the leading cause of visual impairment in the elderly in the developed countries. Intravitreal antivascular endothelial growth factor (VEGF) drugs are currently considered as the first-line treatment option for treating w-AMD; however, the frequent injection intervals have lit the way to investigate novel anti-VEGF agents allowing a more extended treatment regimen. Brolucizumab is a single-chain antibody fragment targeting all the isoforms of VEGF-A. Phase III HAWK and HARRIER trials have shown a longer durability and superior anatomical outcomes as compared with the standard of care by adopting a quarterly regimen for treating w-AMD. Brolucizumab has been approved in Europe, USA, and Japan for the management of w-AMD. AREAS COVERED: This article presents an overview of w-AMD and investigates the progress of brolucizumab through clinical trials. It offers insights into where brolucizumab may be placed in the current market of anti-VEGF agents and its potential advantages over the previous molecules adopted for treating w-AMD. EXPERT OPINION: The possibility of administering brolucizumab with more dilated treatment intervals represents an important advantage to decrease the treatment burden and improve patient compliance. Brolucizumab represents a possible drug switching option in non-responding patients to other anti-VEGF drugs.

Toward the Discovery and Development of PSMA Targeted Inhibitors for Nuclear Medicine Applications.

BACKGROUND: The rising incidence rate of prostate cancer (PCa) has promoted the development of new diagnostic and therapeutic radiopharmaceuticals during the last decades. Promising improvements have been achieved in clinical practice using prostate specific membrane antigen (PSMA) labeled agents, including specific antibodies and small molecular weight inhibitors. Focusing on molecular docking studies, this review aims to highlight the progress in the design of PSMA targeted agents for a potential use in nuclear medicine. RESULTS: Although the first development of radiopharmaceuticals able to specifically recognize PSMA was exclusively oriented to macromolecule protein structure such as radiolabeled monoclonal antibodies and derivatives, the isolation of the crystal structure of PSMA served as the trigger for the synthesis and the further evaluation of a variety of low molecular weight inhibitors. Among the nuclear imaging probes and radiotherapeutics that have been developed and tested till today, labeled Glutamate-ureido inhibitors are the most prevalent PSMA-targeting agents for nuclear medicine applications. CONCLUSION: PSMA represents for researchers the most attractive target for the detection and treatment of patients affected by PCa using nuclear medicine modalities. [99mTc]MIP-1404 is considered the tracer of choice for SPECT imaging and [68Ga]PSMA-11 is the leading diagnostic for PET imaging by general consensus. [18F]DCFPyL and [18F]PSMA-1007 are clearly the emerging PET PSMA candidates for their great potential for a widespread commercial distribution. After paving the way with new imaging tools, academic and industrial R&Ds are now focusing on the development of PSMA inhibitors labeled with alpha or beta minus emitters for a theragnostic application.

The Effect of Antibody Fragments on CD25 Targeted Regulatory T Cell Near-Infrared Photoimmunotherapy.

Regulatory T (Treg) cells play a major role in immune suppression permitting tumors to evade immune surveillance. Depletion of intratumoral Treg cells can result in tumor regression. However, systemic depletion of Tregs may also induce autoimmune adverse events. Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cell-specific cancer therapy that locally kills specific cells in the tumor. Antibody-photoabsorber (IRDye700DX) conjugates (APC) are injected and bind to the tumor, and subsequent administration of NIR light to the tumor results in rapid cell death only in targeted cells. CD25-targeted NIR-PIT has been shown to induce spatially selective depletion of tumor-associated Treg cells. In this study, we compared the efficacy of an antibody fragment, anti-CD25-F(ab')2, and a full antibody, anti-CD25-IgG, as agents for NIR-PIT. Tumor-bearing mice were divided into four groups: (1) no treatment; (2) anti-CD25-IgG-IR700 i.v. only; (3) anti-CD25-F(ab')2-IR700 i.v. with NIR light exposure; and (4) anti-CD25-IgG-IR700 i.v. with NIR light exposure. Although both CD25-targeted NIR-PITs resulted in significant tumor growth inhibition, the anti-CD25-F(ab')2-IR700 based NIR-PIT was superior to the anti-CD25-IgG-IR700 NIR-PIT. The anti-CD25-F(ab')2-IR700 demonstrated faster clearance from the body than the anti-CD25-IgG-IR700. Sustained circulation of anti-CD25-IgG-IR700 may block IL-2 binding on the activated effector T-cells decreasing immune response. In conclusion, anti-CD25-F(ab')2 based NIR-PIT was more effective in reducing tumor growth than anti-CD25-IgG based NIR-PIT. Absence of the Fc portion of the APC leads to faster clearance and therefore promotes a superior activated T cell response in tumors.

A promising cancer diagnosis and treatment strategy: targeted cancer therapy and imaging based on antibody fragment.

With the arrival of the precision medicine and personalized treatment era, targeted therapy that improves efficacy and reduces side effects has become the mainstream approach of cancer treatment. Antibody fragments that further enhance penetration and retain the most critical antigen-specific binding functions are considered the focus of research targeting cancer imaging and therapy. Thanks to the superior penetration and rapid blood clearance of antibody fragments, antibody fragment-based imaging agents enable efficient and sensitive imaging of tumour sites. In tumour-targeted therapy, antibody fragments can directly inhibit tumour proliferation and growth, serve as an ideal carrier for delivery of anti-tumour drugs, or manipulate the immune system to eliminate tumour cells. In this review, the excellent physicochemical properties and the basic structure of antibody fragments are expressly depicted depicted, the progress of antibody fragments in cancer therapy and imaging are thoroughly summarized, and the future development of antibody fragments is predicted.

A Novel Cell-Penetrating Antibody Fragment Inhibits the DNA Repair Protein RAD51.

DNA damaging chemotherapies are successful in cancer therapy, however, the damage can be reversed by DNA repair mechanisms that may be up-regulated in cancer cells. We hypothesized that inhibiting RAD51, a protein involved in homologous recombination DNA repair, would block DNA repair and restore the effectiveness of DNA damaging chemotherapy. We used phage-display to generate a novel synthetic antibody fragment that bound human RAD51 with high affinity (KD = 8.1 nM) and inhibited RAD51 ssDNA binding in vitro. As RAD51 is an intracellular target, we created a corresponding intrabody fragment that caused a strong growth inhibitory phenotype on human cells in culture. We then used a novel cell-penetrating peptide "iPTD" fusion to generate a therapeutically relevant antibody fragment that effectively entered living cells and enhanced the cell-killing effect of a DNA alkylating agent. The iPTD may be similarly useful as a cell-penetrating peptide for other antibody fragments and open the door to numerous intracellular targets previously off-limits in living cells.

Fusion of thymosin alpha 1 with mutant IgG1 CH3 prolongs half-life and enhances antitumor effects in vivo.

Thymosin alpha 1 (Tα1) is an immunomodulatory polypeptide secreted from the thymus. Tα1 has a wide range of biological functions, such as immunomodulation and endocrine regulation. Tα1 also displays antiviral and antitumor activities. Tα1 has been successfully used in clinical adjuvant therapy for solid tumors to improve the immune response of patients undergoing chemotherapy and radiotherapy. However, the half-life of Tα1 in the body is short, so frequent administration is required to maintain efficacy. In order to improve the pharmacokinetic profile of Tα1, we linked the mutated CH3 (mCH3) fragment of IgG1 (human) to the C-terminus of Tα1 to produce a long-acting fusion protein, Tα1-mCH3. The half-life of Tα1-mCH3 (47 h) was substantially increased compared with that of the parent molecule Tα1 (3 h). In vivo studies indicated that mCH3 fusion retained the original biological activity of Tα1, and Tα1-mCH3 showed slightly better immunomodulatory effect than Ta1. In the 4 T1 and B16F10 tumor xenograft models, Tα1-mCH3 induced a greater abundance of CD4+ and CD8+ T-cells in tumor tissues compared with Ta1. Tα1-mCH3 exhibited better effect in promoting the production of IL-2 and IFN-γ compared with Tα1. Therefore, Tα1-mCH3 more efficiently inhibited the growth of 4 T1 and B16F10 tumors than Tα1. In conclusion, fusion with mCH3 is an attractive strategy to lengthen the half-life and increase the activity of Tα1.

[Antibody alternative formats: antibody fragments and new frameworks]. / Les formats alternatifs aux anticorps - Fragments et nouvelles charpentes.

Antibodies are now recognized as routine molecules in many therapeutic fields, no longer restricted to oncology and inflammation. This explosion of the field leads to new needs that can be better fulfilled by molecules inspired but different from conventional antibodies. In particular, the antibody molecule has multiple functions that are not always necessary, such as its ability to recruit immune system cells, its bivalency, or its high plasma half-life. However, in most applications, its remarkable ability to recognize almost any molecular partner with high affinity and specificity must be preserved. In addition, antibodies are very large molecules, expensive to produce and having limited physicochemical properties that limit their use in aggressive media. Finally, in certain therapeutic applications, the large size of the antibody molecule may also limit its diffusion in tissues and prevent the recognition of some poorly accessible molecular structures. To address these limitations, many alternative formats to whole antibodies have been developed over the last twenty years. These new formats have found applications in many fields like biotechnology, in vitro and in vivo diagnosis, and therapy. Two large families of molecules cover this field and will be presented in this mini-review. The first family is based on antibody by reducing its size, such as classical antibody fragments (Fab, scFv) or those derived from camels or sharks (VHH, V-NAR). The second family was developed by first identifying frameworks fulfilling the desired properties, in particular the stability in extreme medium and the productivity in simple and economic systems like bacteria, then by grafting binding properties comparable to antibodies using methods based on in vitro directed molecular evolution techniques. This mini-review will focus on the most advanced molecules but the field is quickly evolving. It should be noted that many of these molecules, or even these approaches, are covered by patents and are often developed by young innovative companies, some of which have been already bought by large pharmaceutical groups.

TITLE: Les formats alternatifs aux anticorps - Fragments et nouvelles charpentes. ABSTRACT: Les anticorps sont désormais devenus d'une utilisation courante dans un large champ thérapeutique qui n'est plus restreint à la cancérologie et à l'inflammation. Cette explosion du domaine conduit à des besoins nouveaux qui peuvent être mieux remplis par des molécules inspirées mais différentes des anticorps classiques. En particulier, la molécule anticorps a de multiples fonctions qui ne sont pas toujours nécessaires, comme sa capacité à recruter les cellules du système immunitaire, à se lier de façon bivalente à sa cible ou à présenter une demi-vie plasmatique élevée. En revanche, dans la grande majorité des applications, sa remarquable capacité à reconnaître spécifiquement sa cible moléculaire et surtout sa diversité de reconnaissance doivent être conservées. De plus, les anticorps sont des molécules de très haut poids moléculaire, coûteuses à produire et qui présentent des propriétés physicochimiques limitées ne permettant pas leur utilisation dans des milieux agressifs. Finalement, dans certaines applications thérapeutiques, la grande taille de la molécule (environ 150 kDa) peut également limiter sa diffusion dans les tissus et empêcher la reconnaissance de certaines structures moléculaires peu accessibles. Pour répondre à ces limitations, de nombreux formats alternatifs aux anticorps entiers ont été développés au cours de ces vingt dernières années. Les applications couvrent les domaines de la biotechnologie, du diagnostic in vitro et in vivo et de la thérapie. Deux grandes familles de molécules permettent de couvrir ce champ et seront présentées dans cette mini-revue. Une première famille s'appuie sur la diversité naturelle des anticorps mais en en réduisant la taille, comme les fragments d'anticorps classiques (Fab, scFv) ou ceux provenant des camélidés ou des requins (VHH, V-NAR). La deuxième famille a été développée en partant des propriétés finales désirées et notamment la stabilité en milieu extrême et la productivité en système simple et économique de production comme l'utilisation de bactéries et en y greffant des propriétés de liaison comparables aux anticorps par des méthodes d'évolution moléculaire dirigée in vitro. Cette mini-revue se concentrera sur les molécules les plus avancées, mais le domaine est en très forte et rapide expansion. Il faut noter que beaucoup de ces molécules, voire ces approches, sont couvertes par des brevets et sont souvent développées dans le cadre de jeunes sociétés innovantes dont certaines ont déjà été rachetées par de grands groupes de la pharmacie.

[Developability assessment]. / Développabilité.

The therapeutic antibodies and their by-products (antibody fragments, conjugated, etc.) establish one of the most dynamic biopharmaceutical market segments today. Due to their intrinsic properties of specificity towards their target, towards their flexible affinity and due to their stability, antibodies became therapeutic agents of the very first choice. One of the challenges of this sector is to create antibodies of very good quality, more and more quickly, while having less and less consequent development costs in fine.

TITLE: Développabilité. ABSTRACT: Les anticorps thérapeutiques et leurs dérivés (fragments d'anticorps, conjugués, etc.) constituent aujourd'hui l'un des segments du marché biopharmaceutique les plus dynamiques. De par leurs propriétés intrinsèques de spécificité vis-à-vis de leur cible, de leur affinité modulable et de par leur stabilité, les anticorps sont devenus des agents thérapeutiques de tout premier choix. Un des challenges de ce secteur est de créer des anticorps de très bonne qualité, de plus en plus rapidement, tout en ayant des coûts de développement de moins en moins conséquents in fine.

Antibody Fragments as Potential Biopharmaceuticals for Cancer Therapy: Success and Limitations.

Monoclonal antibodies (mAbs) are an important class of therapeutic agents approved for the therapy of many types of malignancies. However, in certain cases applications of conventional mAbs have several limitations in anticancer immunotherapy. These limitations include insufficient efficacy and adverse effects. The antigen-binding fragments of antibodies have a considerable potential to overcome the disadvantages of conventional mAbs, such as poor penetration into solid tumors and Fc-mediated bystander activation of the immune system. Fragments of antibodies retain antigen specificity and part of functional properties of conventional mAbs and at the same time have much better penetration into the tumors and a greatly reduced level of adverse effects. Recent advantages in antibody engineering allowed to produce different types of antibody fragments with improved structure and properties for efficient elimination of tumor cells. These molecules opened up new perspectives for anticancer therapy. Here, we will overview the structural features of the various types of antibody fragments and their applications for anticancer therapy as separate molecules and as part of complex conjugates or structures. Mechanisms of antitumor action of antibody fragments as well as their advantages and disadvantages for clinical application will be discussed in this review.

ImmunoPET of Malignant and Normal B Cells with 89Zr- and 124I-Labeled Obinutuzumab Antibody Fragments Reveals Differential CD20 Internalization In Vivo.

Purpose: The B-cell antigen CD20 provides a target for antibody-based positron emission tomography (immunoPET). We engineered antibody fragments targeting human CD20 and studied their potential as immunoPET tracers in transgenic mice (huCD20TM) and in a murine lymphoma model expressing human CD20.Experimental Design: Anti-CD20 cys-diabody (cDb) and cys-minibody (cMb) based on rituximab and obinutuzumab (GA101) were radioiodinated and used for immunoPET imaging of a murine lymphoma model. Pairwise comparison of obinutuzumab-based antibody fragments labeled with residualizing (89Zr) versus non-residualizing (124I) radionuclides by region of interest analysis of serial PET images was conducted both in the murine lymphoma model and in huCD20TM to assess antigen modulation in vivoResults:124I-GAcDb and 124I-GAcMb produced high-contrast immunoPET images of B-cell lymphoma and outperformed the respective rituximab-based tracers. ImmunoPET imaging of huCD20TM showed specific uptake in lymphoid tissues. The use of the radiometal 89Zr as alternative label for GAcDb and GAcMb yielded greater target-specific uptake and retention compared with 124I-labeled tracers. Pairwise comparison of 89Zr- and 124I-labeled GAcDb and GAcMb allowed assessment of in vivo internalization of CD20/antibody complexes and revealed that CD20 internalization differs between malignant and endogenous B cells.Conclusions: These obinutuzumab-based PET tracers have the ability to noninvasively and quantitatively monitor CD20-expression and have revealed insights into CD20 internalization upon antibody binding in vivo Because they are based on a humanized mAb they have the potential for direct clinical translation and could improve patient selection for targeted therapy, dosimetry prior to radioimmunotherapy, and prediction of response to therapy. Clin Cancer Res; 23(23); 7242-52. ©2017 AACR.

Predicting the response to CTLA-4 blockade by longitudinal noninvasive monitoring of CD8 T cells.

Immunotherapy using checkpoint-blocking antibodies against targets such as CTLA-4 and PD-1 can cure melanoma and non-small cell lung cancer in a subset of patients. The presence of CD8 T cells in the tumor correlates with improved survival. We show that immuno-positron emission tomography (immuno-PET) can visualize tumors by detecting infiltrating lymphocytes and, through longitudinal observation of individual animals, distinguish responding tumors from those that do not respond to therapy. We used 89Zr-labeled PEGylated single-domain antibody fragments (VHHs) specific for CD8 to track the presence of intratumoral CD8+ T cells in the immunotherapy-susceptible B16 melanoma model in response to checkpoint blockade. A 89Zr-labeled PEGylated anti-CD8 VHH detected thymus and secondary lymphoid structures as well as intratumoral CD8 T cells. Animals that responded to CTLA-4 therapy showed a homogeneous distribution of the anti-CD8 PET signal throughout the tumor, whereas more heterogeneous infiltration of CD8 T cells correlated with faster tumor growth and worse responses. To support the validity of these observations, we used two different transplantable breast cancer models, yielding results that conformed with predictions based on the antimelanoma response. It may thus be possible to use immuno-PET and monitor antitumor immune responses as a prognostic tool to predict patient responses to checkpoint therapies.