Highly specific rabbit single-domain antibodies for drug delivery in immunotherapy applications

Abstract

The present disclosure relates to the development of drug delivery systems comprising single domain antibodies (sdAbs). To this end, antibody-drug conjugates (ADCs) are provided that exhibit high selectivity and efficiency to be advantageously used in cancer therapy, as well as drug delivery systems that target central nervous system (CNS) pathologies. The ADC molecules developed for therapy, i.e., cancer therapy, are V L The drug delivery system developed for targeting BBB endothelial cell receptors in the central nervous system (CNS) comprises rabbit-derived single domain antibodies (sdAbs) conjugated to the surface of liposomes encapsulating suitable drugs that enable efficient blood-brain barrier (BBB) ​​translocation. The respective production methods are also disclosed herein. Thus, the present disclosure is in the field of genetic engineering, biotechnology, medicine and medicine.

Description

[Technical field]

[0001] The present invention relates to the development of a drug delivery system comprising a single domain antibody (sdAb).

[0002] To this end, antibody-drug conjugates (ADCs) are provided that exhibit high selectivity and efficacy for advantageous use in cancer therapy, as well as drugs targeting central nervous system (CNS) pathologies.

[0003] ADC molecules developed for cancer therapy, i.e., V L It is derived from a rabbit-derived sdAb that contains a potent cytotoxic payload, the SN38 small molecule, conjugated to the free exposed cysteine ​​at position 80 of the framework.

[0004] Methods for obtaining such antibody fragments and their use as drugs are also disclosed. The method for obtaining such molecules involves selective conjugation of free cysteines present in rabbit-derived sdAbs with chemical payloads without the need for further genetic engineering.

[0005] A drug delivery system developed to target blood-brain barrier (BBB) ​​endothelial cell receptors in the central nervous system (CNS) comprises rabbit-derived single domain antibodies (sdAbs) conjugated to the surface of liposomes encapsulating appropriate drugs to enable efficient BBB translocation. Methods for the production of each are also disclosed herein.

[0006] Thus, the present invention is in the fields of genetic engineering, biotechnology, pharmaceuticals and medicine. [Background technology]

[0007] Cancer morbidity and mortality continues to grow worldwide, causing nearly 10 million deaths and 19.3 million new cases in 2020 alone. Due to rapid population growth and aging, as well as the increasing prevalence of risk factors, the number of new cancer cases is expected to rise by 47% over the next 20 years. Thus, despite the significant progress made in cancer treatment over the last decade, there remains a great demand for new solutions.

[0008] Although nearly 80 years have passed since the advent of modern cancer chemotherapy, traditional chemotherapy has changed little and still uses cytotoxic drugs that target the cell cycle of rapidly dividing cancer cells. Although it is associated with significant clinical drawbacks, including a narrow therapeutic window, increasing drug resistance, and nonspecific toxicity, cytotoxic chemotherapy remains at the heart of cancer treatment.

[0009] In the 1970s, the advent of monoclonal antibody (mAb)-based therapy was expected to revolutionize cancer treatment. By specifically targeting cancer cells, mAbs reduce nonspecific toxicity, directly promote signal transduction-induced cell death, or mediate antitumor immune responses. To date, about 30 mAbs have been approved by the US Food and Drug Administration (FDA) for cancer treatment, but many mAbs do not have clinical efficacy as single agents and are currently used in combination with traditional cytotoxic regimens.

[0010] Advances in mAb technology over the following decades allowed antibodies to be conjugated with a variety of antitumor effector molecules (e.g., cytotoxic drugs, radiopharmaceuticals, and immunotoxins), driving the development of mAb-based targeted and immunotherapies, including an emerging class of anticancer therapeutics called antibody-drug conjugates (ADCs). ADCs combine the tumor selectivity, pharmacokinetics, and biodistribution properties of antibodies with the cytotoxic potency of small molecules. This concept of selective delivery was first envisioned in the early 20th century by Paul Ehrlich, who discussed the "magic bullet" theory and revisited it when mAbs were considered a suitable part for the creation of such magic bullets.

[0011] Nevertheless, the road to developing effective ADCs has proved to be long and extremely difficult in itself. First generation ADCs, consisting of mAbs conjugated with traditional chemotherapeutic agents, had limited success due to low efficacy and / or toxicity associated with ADC instability and systemic loss of the drug. Thus, next generation ADCs with more potent payloads relied on humanized and human mAbs to optimize linker stability and intracellular release, and reduce immunogenicity while increasing target and antibody selectivity. This led to the FDA approval of the first ADC, gemtuzumab ozogamicin, in 2000 for the treatment of CD33-expressing acute myeloid leukemia (AML).

[0012] Since then, a remarkable expansion of the clinical ADC pipeline has occurred, with over 80 ADCs, enrolled in about 600 clinical trials at different clinical stages to date. However, only 9 additional ADCs have been granted FDA approval since 2011. Over the years, several ADCs showing great potential in early preclinical stages have failed to progress or even been abruptly terminated. To date, about 55 ADC clinical trials have been discontinued, mostly due to lack of efficacy and off-target cytotoxicity. Therefore, a careful and critical reevaluation of preclinical and clinical results is essential to inform future trials and enable the success of this promising platform.

[0013] One of the main challenges affecting ADCs, including those already on the market, is the heterogeneous composition of the resulting products, meaning that each mAb is linked to a variable number of cytotoxic drugs at different positions. This heterogeneity results in different drug-to-antibody ratios (DARs), generating products with variable pharmacokinetic and therapeutic profiles. This issue is mostly associated with traditional drug bioconjugation methods that conjugate antibodies either through surface-exposed lysines (approximately 70-90) or cysteines derived from interchain disulfides (eight in IgG1), two abundant features in IgG mAbs.

[0014] Moreover, conserved cysteines play a fundamental role in antibody structure, and their use in conjugation often leads to aggregation problems and improper folding.New approaches have been used to overcome these shortcomings, including site-specific conjugation methods that have led to a new generation of more uniform, molecularly defined ADCs.However, many of these methods are not compatible with the scale-up of manufacturing methods required for ADC production.

[0015] Moreover, many ADCs currently in development and on the market are composed of complete IgG antibodies. However, the clinical use of these IgG-based moieties has been hindered by their poor penetration into tumor tissues as a result of their large molecular weight, and by the high manufacturing costs in mammalian cells. Furthermore, there is now evidence that the Fc domain of IgG may be unnecessary or even undesirable for the efficacy of ADCs. In fact, the long half-life of ADCs promoted by FcRn increases their exposure to healthy tissues, while FcγR cross-reacts with endothelial cells and immune cells, both biological processes associated with off-target toxicity.

[0016] Therefore, further improvements in ADC design and development are needed to enable the synthesis of more homogeneous and stable molecules with higher therapeutic indices.

[0017] To overcome these problems, the present invention provides a drug delivery system based on rabbit-derived single domain antibodies (sdAbs). sdAbs are currently the smallest functional antibody fragments, consisting of only VH or VL units. These small-sized scaffolds of approximately 15 kDa have higher tumor penetration and accessibility to targets that are not easily reached by the larger size of conventional mAbs.

[0018] As mentioned above, a second aspect of the present invention is to provide a drug delivery system based on rabbit-derived single domain antibodies (sdAbs) against blood-brain barrier (BBB) ​​endothelial cell receptors, targeting central nervous system (CNS) pathologies, i.e. capable of crossing the BBB.

[0019] Despite great advances in the fields of neuroscience and drug development, the efficacy of many potential therapeutic agents for the treatment of central nervous system (CNS) diseases has been systematically challenged by the low permeability of the blood-brain barrier (BBB). In fact, this physical and metabolic selective barrier between the brain and the systemic circulation is the main obstacle in brain-targeted drug development and the most important factor limiting the treatment of major unmet neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and brain tumors. To overcome this barrier, several strategies have been elaborated in the past two decades. One such approach is the development of specific antibodies that target endogenous BBB transport mechanisms such as the receptor-mediated transcytosis (RMT) system. By using this natural pathway, antibodies specifically bind to BBB receptors and translocate therapeutic compounds into the brain in a controlled, non-damaging manner as biological "Trojan horses". The potential of this strategy for CNS drug delivery has already been well validated for two targets: insulin receptor (IR) and transferrin receptor (TfR). Antibodies against these two receptors have shown the ability to transport therapeutic drugs across the BBB via RMT, validating the potential of this route for therapy and diagnosis of neurological diseases. However, IR and TfR are not brain-specific but are highly expressed in other tissues and are involved in metabolically important cellular functions. Therefore, antibodies against these receptors may result in mistargeting of brain drugs to other sites, thus resulting in undesirable side effects and creating safety risks. Moreover, the majority of developed antibodies are IgG, a class of macromolecules that limit their accessibility and translocation to the brain, and are unable to reach sufficient concentrations in the brain side systematically, hindering their therapeutic potential. In addition, IgG uptake by widely expressed Fc receptors, together with its long half-life, further contributes to its non-specific accumulation that may result in systemic secondary effects. Therefore, there is an urgent need to identify more selective BBB targets or improved antibodies that can enhance uptake of therapeutic molecules into the brain while minimizing non-specific accumulation.For targeting and drug delivery functions, the only components of IgG molecules that are necessary are the antibody variable binding domains, VH or VL. Their small size allows access to epitopes inaccessible to conventional IgG, and together with the possibility of controlled drug conjugation engineering, paves the way for new drug delivery strategies to successfully transpose the BBB. Furthermore, single domain antibodies (sdAbs) are highly stable moieties, exhibit low immunogenicity, reduced production costs, and their structure allows flexible conjugation to neuropharmaceuticals or nanoparticles containing bioactive compounds, making them very attractive drug delivery vectors.

[0020] More recently, detailed transcriptomic and proteomic analysis of mouse brain endothelial cells, together with translation to the human setting, allowed the identification of three robust BBB receptors for drug delivery: basigin, glutamate receptor 1 and CD98 heavy chain (CD98hc). Antibodies developed against CD98hc exhibited improved brain targeting and drug delivery properties when compared to IR and TfR.

[0021] Nevertheless, these BBB targets were identified by in vitro selection tests that do not completely mimic the in vivo environment of the BBB and may impair the expression level and conformation of their native receptors. Indeed, when we look at the BBB characteristics in vivo, it is known that BBB endothelial cells are stimulated by their surrounding cells and intraluminal blood flow. This regulates the expression of specific receptors on the cell surface in a polarized manner that contributes to the complexity of the BBB. For these reasons, screening for highly selective BBB transmigrate antibodies should preferably be performed in vivo. Summary of the Invention [Problem to be solved by the invention]

[0022] Therefore, to solve these problems of the prior art, the present invention proposes a new approach involving in vivo immunization of whole cells in rabbits followed by in vivo phage display selection in a mouse model, aiming to develop a potent BBB-translocating nanobody scaffold. [Means for solving the problem]

[0023] To achieve this, a rabbit-derived immunized sdAb library against brain endothelial cell receptors was constructed and brain-specific nanobodies were recovered in an in vivo phage display assay.

[0024] Using this combinatorial approach, we successfully identified a panel of novel BBB-crossing sdAbs that can specifically target and reach the brain. To evaluate the ability of our selected sdAbs for CNS drug delivery, a number of promising lead antibodies were modified with the surface of liposomes encapsulating a model drug that has been proven not to cross the BBB, the pan-histone deacetylase inhibitor (HDACi) panobinostat (PAN), and their BEB translocation properties and antitumor activity were evaluated in a dual-functional in vitro BEB-glioblastoma model.

[0025] Definitions and Abbreviations aa amino acid ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) ADCC antibody-dependent cytotoxicity Amp Ampicillin Anti-HA-HRP Anti-hemagglutinin-horseradish peroxidase BM Bone marrow BSA Bovine Serum Albumin C Carboxyl terminal CDC Complement-dependent cytotoxicity cDNA Complementary DNA CDR Complementarity Determining Region CH heavy chain constant region cNHL Canine Non-Hodgkin's Lymphoma CL light chain constant region DMEM Dulbecco's Modified Eagle's Medium DNA deoxyribonucleic acid E.coli Escherichia coli ELISA Enzyme-Linked Immunosorbent Assay Fab fragment antigen binding Fc constant fragment FDA Food and Drug Administration LB Luria-Bertani medium mAb Monoclonal antibody N-amino terminus NHL Non-Hodgkin's Lymphoma NK cells Natural killer OD optical density PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction PEG-8000 Polyethylene glycol pT7-PL pT7-peptide leader RNA Ribonucleic acid RPM Revolutions per minute RPMI Roswell Park Memorial Institute Medium RT Room temperature (20~25℃) SB Super Medium scFv single chain variable fragment sdAb Single Domain Antibody SOC Super Optimal Bros. SP Spleen UV ultraviolet light VH Heavy chain variable domain VL light chain variable domain [Brief description of the drawings]

[0026] [Figure 1]Characterization of rabbit immune responses. Rabbit immunization: Three female New Zealand white rabbits were immunized with 1x107 cNHL primary cells from our biobank over a period of four months. cNHL primary cells were selected from patients diagnosed with DLBCL. Five days after the last boost, rabbits were sacrificed and spleen and bone marrow were harvested for total RNA isolation and cDNA synthesis. Serum titration by ELISA. 5x104 cells were incubated with serial dilutions (1 / 1000 to 1 / 32000) of rabbit serum. All three rabbit samples exhibited high responses against cNHL primary cells and the CLBL-1 cell line. [Figure 2A] ~ [Figure 2B] FIG. 1 shows FACS analysis confirming the results obtained by ELISA. [Diagram 3] Figure 1 shows protein profile analysis of rabbit serum by immunoblotting of CLBL-1, cNHL primary cells and PBMCs derived from healthy dogs. The results showed potential tumor-specific epitopes recognized on CLBL-1 and cNHL primary extracts. Representative blots are shown. [Figure 4] Figure 4. Phage display selection in vitro and in vivo. To select the best antibodies for non-Hodgkin's lymphoma (NHL) target, a previously constructed sdAb immune library with a diversity of 10 was used for in vitro whole cell and in vivo phage display in a cNHL xenograft mouse model. Figure 4A - First, whole cell phage display was performed. The number of washes was increased over the selection to improve stringency and recover phage clones with higher affinity and specificity. Two different elution methods were performed to recover binders and internalized antibodies. Figure 4B - After in vitro selection, a final in vivo phage display was performed in a mouse xenograft cNHL model. Briefly, phage output from the third round of in vitro panning was collected, re-amplified, and tail vein injected into a xenograft mouse cNHL model. After 60 min, the mice were euthanized and phage were recovered from the tumor. [Diagram 5] Figure 5 shows the results of selection with fewer phages in the output titer, showing high enrichment for phages targeting cNHL. Figure 5A shows the results obtained for VH library screening. Figure 5B shows the results obtained for VL library screening. [Figure 6] Screening for NHL targets. Figure 6A - To determine the best lead candidates, approximately 200 clones were tested in an ELISA assay. Three parameters were evaluated: binding, expression and non-specific binding. Those that showed stronger signals to NHL cell extracts were selected. Figure 6B - The selected 43 best clones were analyzed by Sanger sequencing. [Figure 7] To more fully characterize the sequences enriched during in vivo phage display, next generation sequencing was performed. Two samples were sequenced: biopanning and initial immune libraries from cNHL and hNHL tumor models. [Figure 8]Figure 1: Binding and internalization characterization of C5 VLsdAb. C5 binding and internalization properties to CLBL-1 cells were evaluated by cytometry and immunofluorescence. A) For flow cytometry analysis, 1x106 CLBL-1 cells were incubated with Live / Dead reagent for 30 min. 3 μM C5 was then incubated with the cells at 37°C for various time points. Cells were then washed, fixed, permeabilized, incubated with anti-HA antibody for 30 min, washed twice, and incubated with anti-rat Alexa Fluor-488. C5 was shown to bind to CLBL-1 cells. B) In contrast, no binding of C5 was detected for Jurkat cells. C) To confirm the binding of C5 to CLBL-1 verified by flow cytometry, we evaluated its distribution on cells by immunofluorescence assay. A high density of Alexa Fluor-488 labeled C5 can be observed labeled in the perinuclear region. D) In ​​contrast, there is no detectable fluorescence in either the control images or in the presence of Jurkat cells. Representative photomicrographs are shown with C5 (green) and DAPI stained nuclei (blue). [Figure 9] Figure 2 shows the biodistribution profile of 99TmTc-C5. To evaluate the tumor uptake and pharmacokinetic profile of C5 VL sdAb, biodistribution assays were performed on a xenograft model of cNHL at two different time points (15 min and 3 h). C5 was radiolabeled with 99mTc(CO)3(H2O)3 and injected intravenously into the tail vein of a xenograft mouse model of cNHL. Mice were sacrificed at 15 min and 3 h and radioactivity in each organ was measured. Activity in each organ was calculated and expressed as a percentage of the injected radioactive dose per gram of organ or tissue (%ID / g). A) Results show that tumor uptake was approximately 1.5±0.5%ID / g at 15 min and decreased to 1.5% 3 h after injection. Rapid elimination in major organs was found except for liver and spleen. C) Results were also confirmed by Western blot analysis. Representative blots are shown. [Figure 10]Figure 2: Biodistribution of C5-DAB-SN-38. The conjugate C5-DAB-SN-38 was obtained from C5 cysteine ​​modification in PBS buffer at pH 7.4 (10 μM) at room temperature. Top right: Ribbon representation of the predicted 3D structure of C5 sdAb. CDR domains are highlighted in purple (CDR1), green (CDR2) and blue (CDR3). Side chain atoms are shown in stick representation and colored yellow. Van der Waals surfaces are depicted in transparent color. Surfaces of CDR3 and Cys are highlighted. Numbering of CDRs and amino acids was done according to Kabat et al. Bottom right: A) High-resolution mass spectrum of conjugate C5-DAB-SN-38. B) Deconvoluted high-resolution mass spectrum of C5 (12879.2 Da). C) Deconvoluted high-resolution mass spectrum of conjugate C5-DAB-SN-38 (13651.5 Da). [Figure 11]Figure 1 shows the cytotoxic effect of C5-DAB-SN-38. To determine the effect of C5-DAB-SN-38 on CLBL-1 and Jurkat cells, cell viability assays were performed using WST-1 reagent. 6x104 cells were seeded and treated with increasing amounts of compound. After 48 hours of treatment, cell viability was evaluated. A) C5-DAB-SN-38 showed a dose-dependent toxic effect on cNHL cells. B) On the other hand, ADC had no effect on Jurkat cells, proving the specificity of ADC. C5 was used as a control. Best-fit EC50 values ​​for each formulation were calculated using GraphPad Prism software (version 9.2.0, San Diego, CA, USA) using the logarithm (inhibitor) vs. response (variable slope) function. D) Effect of C5-DAB-SN-38 on DNA TopoI activity. To evaluate the effect of C5-DAB-SN-38 on DNA TopoI, we used a human topoisomerase I assay kit to assess its activity. C5-DAB-SN-38 was incubated with 1× reaction buffer and 10 U of Topo I for 1 hour at 37° C., followed by the addition of supercoiled DNA. To stop the reaction, stop loading buffer was added. DNA TopoI activity was visualized by 1% agarose gel electrophoresis obtained with ethidium bromide. Relaxed DNA showed TopoI activity, whereas supercoiled DNA showed inhibition. The presence of higher concentrations of C5-DAB-SN-38 inhibited DNA TopoI in a dose-dependent manner, highlighting the effect of SN-38, present on the ADC, as a cause of cell death. Relaxed and supercoiled DNA were used as positive / negative controls. C5 and SN-38 were also used as controls. [Figure 12] Schematic representation of the in vivo screening method for selecting highly specific BBB-crossing sdAbs. [Figure 13]Figure 13 shows the analysis of rabbit immune response. Figure 13A shows the ELISA serum titration of whole sera from rabbit #1 and rabbit #2 against bEnd.3 endothelial cells. Whole rabbit sera were diluted by serial dilution (500-32,000-fold) and tested at 2 x 104 cells / well. The results showed selective and specific immune response against bEnd.3 cells. Figure 13B shows the target validation and evaluation of potential bEnd.3 receptors recognized by each rabbit serum after immune boost by WB. Total protein extracts were obtained from bEnd.3 using RIPA buffer, and 10 μg and 20 μg were loaded into 11% SDSpage acrylamide gel. After gel transfer and membrane blocking, 500-fold diluted whole rabbit serum was added to determine the specificity of antibodies generated by rabbit immunization method against bEnd.3 whole protein extract. The results showed that antibodies against several receptors were generated in both immunized rabbits validating our immunization method. [Figure 14]Figure 14A: Validation of the BBB-crossing properties of rabbit serum. To confirm that the antibodies obtained from the rabbit immunization method retained the ability to cross the BBB, purified serum from both rabbits was tested in both in vitro and in vivo models. For the in vitro BEB model, 4x103 bEnd.3 cells were seeded in tissue culture inserts of 24-well plates and incubated for 11-14 days in a humidified chamber at 37°C with 5% CO2 added. Figure 14A: To determine the translocation efficiency of rabbit serum, 10μg of purified serum was added to the top, incubated for 15 and 60 minutes, and collected from both the top and bottom and analyzed by WB. Rabbit serum was detected at the bottom of the single-cell model at both time points validating the translocation ability of rabbit IgG. To confirm the ability to reach the brain in the in vivo model, purified serum (250μg) was injected intravenously into the tail vein, and after 2 and 60 minutes, the mice were sacrificed and the brains were extracted. Figure 14B: Rabbit immunoglobulins were recovered from mouse serum, homogenized brains by IP with Protein A beads, and analyzed by WB with goat anti-rabbit HRP IgG at 1:1000. Detection of rabbit serum in brain extracts verified the ability of rabbit-derived antibodies to reach mouse brain and further confirmed the expected species cross-reactivity. [Figure 15]Figure 1. Selection of sdAbs by in vivo phage display and NGS. To select the most promising sdAbs for brain targeting and BBB translocation, rabbit-derived immune phage display libraries were injected intravenously into the tail vein of CD1 mice. Phages were allowed to circulate for 2 and 60 min, mice were perfused and sacrificed, and phages extracted from the brain were re-amplified. Three rounds of in vivo biopanning were performed, phages recovered from the brain were re-amplified and re-injected into mice for a new selection round. For each selection round, quantification of injected phages (input) and recovered phages (output) from the brain was determined. After in vivo selection, the selected phage library was re-amplified and analyzed by next generation sequencing, and data was analyzed by in-house bioinformatics scripts. Sequences are presented with more than 20 representatives at at least one time point. Selected clones for in vivo biodistribution after expression and stability assays are depicted. [Figure 16] Figure 16. In vitro selection of dominant sdAb clones. To select the clones most qualified for crossing the BBB, clones showing higher frequency in NGS analysis and superior in terms of expression and solubility properties (data not shown) were selected for further characterization in vitro for BEB model translocation efficacy. Figure 16A: 15 μg each of purified sdAb was added to the top of the transwell and incubated for 90 min. Figure 16B: After the incubation period, the entire volume was collected from the top and bottom and analyzed by WB with anti-HA antibody. [Figure 17]Figure 17 shows the translocation of RG3 and FC5 functionalized liposomes (Lip-RG3 and Lip-FC5) in an in vitro BEB model. To determine the effect of RG3 functionalization in liposomes on in vitro BEB translocation, rhodamine-loaded, biotinylated liposomes were surface-modified with RG3 as previously described, added to the top of transwells, and incubated for 90 min (Figure 17A), 6 h (Figure 17B), and 24 h (Figure 17C). Translocation percentage was determined by measuring rhodamine fluorescence intensity at the bottom. Non-functionalized liposomes (Lip) and FC5 functionalized liposomes (Lip-FC5) were used as controls. One-way ANOVA followed by Turkey's test was used to compare each liposomal formulation (****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not statistically significant). [Figure 18]An in vitro BEB-GBM model was developed to mimic the complexities of drug delivery to brain tumors. Figure 18 shows the activity of liposomal drug delivery in the BEB-glioblastoma model. The model combines a monolayer of bEnd.3 as a BBB barrier and LN229 cell line at the bottom as a glioblastoma tumor. Figure 18A shows the ability of free PAN to translocate the BBB and the evaluation of its effect on the integrity of the model barrier. PAN was added at increasing concentrations to the top of the BEB-GBM model and incubated for 24 hours. The integrity of bEnd.3 was measured by determining the translocation of FD40 fluorescent probe, while the viability of LN229 was determined using WST-1 reagent and OD450nm measurements after 24 hours of incubation. Figure 18B: RG3 and FC5 functionalized liposomes encapsulating PAN were analyzed for BEB translocation and delivery of PAN to glioblastoma cell lines, and compared with non-functionalized, unloaded liposomes (control). Briefly, PAN-loaded liposomes were added to the top of the BEB-GBM model and incubated for 24 hours. The cytotoxic effect of PAN in LN229 was determined by adding WST-1 reagent. Values ​​were obtained from two replicates of two independent experiments (Figure 7B1, Figure 7B2). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present invention relates to the development of antibody fragments, such as single domain antibodies (sdAbs), as alternative targeting agents for drug delivery systems.

[0028] To this end, antibody-drug conjugates (ADCs) have been developed that exhibit high selectivity and efficacy for advantageous use in cancer therapy and central nervous system (CNS) pathologies.

[0029] In one aspect, the present invention provides a method for producing a medicament for the treatment of a cancer, comprising: LThe present invention relates to an ADC molecule developed for therapy, namely cancer therapy, comprising a rabbit-derived sdAb conjugated to the free exposed cysteine ​​at position 80 of the framework and a potent cytotoxic payload.

[0030] Currently, sdAbs are the smallest functional antibody fragments, consisting of only VH or VL units. These small size scaffolds of about 15 kDa have higher tumor penetration and accessibility to targets not easily reached by the larger size of conventional mAbs. Furthermore, their fast clearance rate compared to intact IgG may be advantageous when the risk of toxicity in healthy tissues increases with long-term exposure.

[0031] In addition to their reduced size, sdAbs also offer greater stability, solubility, less immunogenicity and lower production costs since they can be expressed in bacterial systems. Importantly, rabbit-derived V L The sdAb has a free exposed cysteine ​​at position 80 in its native framework region that can be exploited for selective conjugation of chemical payloads without the need for further genetic engineering.

[0032] In the above results, the ADC molecules of the invention contain a payload potent enough to kill the target cancer cells, the SN38 small molecule conjugated to a free cysteine.

[0033] Methods for obtaining such ADCs and their use as drugs are also disclosed. The method for obtaining such molecules involves selective conjugation of free cysteines present in rabbit-derived sdAbs with chemical payloads without the need for further genetic engineering.

[0034] In another aspect, the present invention relates to molecules specifically developed for central nervous system (CNS) therapy that are capable of crossing the blood-brain barrier (BBB). These drugs also include rabbit-derived single domain antibodies (sdAbs) that specifically target BBB endothelial cell receptors.

[0035] Also disclosed are methods for obtaining these molecules by using constructs of rabbit-derived single domain antibody (sdAb) libraries conjugated to the surface of appropriate drug-loaded liposomes that enabled efficient BBB translocation and exhibited potent antitumor activity.

[0036] I. Description of methods for producing antitumor ADCs according to preferred embodiments of the present invention According to a preferred embodiment of the present invention, V L A drug delivery system, VL-DAB-SN38, for use in treatment or tumor cell therapy is provided, which comprises a VL chain modified to display a single free exposed cysteine ​​at position 80 of the framework and modified with the cytotoxic drug SN38 and DAB-SN38, a maleimide-containing molecule, linked via a diazaborine bioconjugation linker.

[0037] The method for obtaining the VL-DAB-SN38 drug delivery system comprises the following main steps: a) providing primary lymph node cells derived from a canine multicentric lymphoma biobank; b) 1 × 10 from the Canine Multicentric Lymphoma Biobank 7 immunizing a rabbit with the lymph node primary cells; c) isolating RNA and cDNA from the spleen and bone marrow samples; d) using the engineered single domain antibody (sdAb) targeting cNHL; e)V L V with a free exposed cysteine ​​at position 80 of the framework L using a rabbit sdAb in the light chain variable region format; f) binding of VL to cNHL cells with incubation of anti-HA FITC antibody; g) bioconjugating VL with DAB-SN38 by adding a solution of DAB-SN38 to a solution of TCEP and VL. It can be described as including:

[0038] In a preferred embodiment, more than 200 clones exhibit anti-tumor properties.

[0039] In a more preferred embodiment, 43 clones exhibited strong signals against non-Hodgkin's lymphoma (NHL) cells (SEQ ID NO: 1 to SEQ ID NO: 43 in the sequence listing).

[0040] Of these, six clones: A12, C8, C5, E1, E2, B12 showed the best results and had the following sequences: SEQ ID NO:1 (clone A12) SEQ ID NO:2 (clone C8) SEQ ID NO:3 (clone C5) SEQ ID NO:4 (Clone E1) SEQ ID NO:5 (Clone E2) SEQ ID NO:6 (clone B12).

[0041] 1. Generation and screening of high-titer antisera against cNHL To develop highly specific cNHL sdAbs, we first generate highly specific sera against cNHL antigens by immunizing selected rabbits with lymph node primary cells derived from a canine multicentric lymphoma from the Faculty of Veterinary Medicine, University of Lisbon.

[0042] Immunization can be monitored by antibody titers, specifically by ELISA and FACS.

[0043] As shown in Figure 1, cell ELISA assays showed that the final sera of all three rabbits exhibited a highly specific and selective response against our biobank cNHL primary cells and the canine B-cell lymphoma cell line, CLBL-1 cell line (Figure 1). Moreover, these data showed that immunization resulted in a strong immune response, showing high serum titers (1:60.000), contrary to the pre-bleed serum. FACS analysis confirmed these results (Figure 2). Immunoblotting was performed to evaluate the protein profile recognized by the rabbit sera. As shown in Figure 3, the protein profiles recognized on cNHL primary and CLBL-1 extracts were similar. On the other hand, various differences were detected between the receptors recognized on PBMCs. The obtained results showed potential tumor-specific epitopes detected by rabbit antibodies present on cNHL cells and revealed the therapeutic potential of these antibodies.

[0044] 2. Construction of Immunized Libraries and Selection of Phage Display To select the most promising antibodies for NHL targeting, we used V-antigens recovered from bone marrow and spleen of selected immunized rabbits, including both cNHL primary cells presenting with Diffuse Large B Cell Lymphoma (DLBCL) from a biobank. L The amplification of V provides or constructs an sdAb library. L The sdAb region was cloned into the pComb3X phagemid vector and used for in vitro and in vivo phage display selection in a mouse model. 11~12 This results in a phage display library with a diversity of 100 bp (Figure 4).

[0045] First, whole-cell phage display is performed to select the most promising sdAbs targeting cNHL epitopes (Figure 4A). This cell phage display screening protocol is based on previous work by Carlos Barbas and the present inventors (Barbas III, CF, Burton, DR, Scott, JK & Silverman, GJ Phage Display: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, 2001) and Dias, JNR et al. Characterization of the canine CD20 as a therapeutic target for comparative passive immunotherapy. Sci Rep 12, 2678 (2022)), which reports a novel whole-cell selection protocol designed to select mAbs that recognize potentially internalizing surface epitopes.

[0046] Over the course of selection, stringency can be improved by increasing the number of washes to collect phage clones with higher target affinity or specificity. Different elution methods can be performed to select for sufficient binders and internalized antibodies.

[0047] As shown in FIG. 5, three rounds of in vitro phage display panning yielded input titers (approximately 10 11~12 ~10 3~4A smaller number of phages in the output titer was obtained compared to the 100% pfu (pfu). Furthermore, the biopanning profile indicates that in vitro phage display successfully led to enrichment of highly specific cNHL sdAbs. After in vitro phage display selection, a final in vivo phage display panning was performed in a mouse xenograft NHL model (Figure 4B). Phage-displayed output recovered from the third round of in vitro panning was collected, re-amplified, and tail vein injected into a xenograft mouse cNHL model previously described by Dias et al. in Dias, JNR et al. Establishment of a bioluminescent canine B-cell lymphoma xenograft model for monitoring tumour progression and treatment response in preclinical studies. PLOS ONE 13, e0208147 (2018). At 60 min post-injection, mice were euthanized, tumors were removed, phages were harvested, and cnhl-specific sdAbs were harvested. By circulating the enriched antibody panel in mice and collecting sdAbs that targeted xenograft tumors, we hoped to select antibodies that fulfill a defined biological effect that would confirm the in vivo availability of the epitope. At the end of this in vivo panning, the recovered phages were collected at 10 6 V L Binder and 10 4 V L This reflected a diversity of internalized factors (phage / Ml) and a high enrichment for phages exhibiting high ability to target cNHL.

[0048] 3. Screening for NHL targets After phage display selection, the best anti-cNHL V LTo express and select sdAbs, phagemid DNA from the in vivo output selection was cloned into the PT7-PL vector and transformed into E. coli strain BL21. Individual clones were then autoinduced and the supernatants were tested in ELISA assays against CLBL-1 and Jurkat cell extracts. To select the best lead candidates, three parameters were evaluated: binding to cNHL, expression yield, and non-specific binding. Approximately 200 clones were screened and V showed stronger signals against CLBL-1. L sdAbs were selected (Figure 6A). After selection, the 43 best lead candidates were analyzed by Sanger sequencing to characterize the selected clones (Figure 6B). To gain further insight into the enriched sequences during in vivo phage display selection, we performed next generation sequencing (NGS) of the biopanning repertoire. NGS allows large-scale sequence analysis of the panning population, allowing genomic assessment of library diversity and frequency of each clone. Two samples: biopanning and initial immune V from the CLBL-1 tumor model were analyzed. LThe sdAb cNHL library was sequenced (Figure 7). Upon bioinformatics analysis of the NGS data, we were able to determine the diversity of each sample and identify the most representative clones. A total of 34880 sequences were obtained for biopanning from CLBL-1 tumors. The cNHL library yielded a total of 48864 sequences. The number of singletons consisting of sequences showing a single occurrence varied between samples, 76.8% in the library and 33.3% in the in vivo biopanning from CLBL-1. Comparison of sequence prevalence between biopanning and library allowed us to verify that the number of occurrences in each sequence was higher in the biopanning clones. These results demonstrated the specificity of the phage display selection, which reduced the high diversity of clones present in the library. By comparing ELISA data, Sanger sequencing and NGS analysis, the six best clones (A12, C8, C5, E1, E2, and B12) were produced and purified. Due to its binding and production properties, the C5 sdAb clone was selected for full characterization and used in the development of the proposed ADC.

[0049] 4. Characterization of Cell Binding by FACS FACS analysis was performed to evaluate the binding of VL C5 to CLBL-1 cells. VL was incubated with cHNL cells for various time points. As shown in Figure 8 (panels A and B), VL was shown to bind to cells and increase its interaction with time. Controls showed no interaction with the antibody.

[0050] 5. Assessing Cell Binding by Immunofluorescence To follow the binding of VL C5 to CLBL-1 cells by FACS analysis, we further evaluate the distribution of the antibody on cells using immunofluorescence assay. As shown in Figure 8 (panels C and D), a high density of VL C5 containing Alexa Fluor-488 can be observed in the perinuclear region. Conversely, there is no detectable fluorescence in the control images. Thus, these images confirmed VL binding to CLBL-1 cells and its internalization into the cytoplasm. This internalization property was essential to develop an effective ADC.

[0051] 6. C5V L Biodistribution study of sdAb Selected C5 V L To evaluate the tumor uptake and pharmacokinetic profile of the sdAb, a biodistribution assay was performed on a xenograft model of cNHL at two different time points (15 min and 3 h). For that purpose, C5 was used as described in the Materials and Methods section. 99m The antibodies were radiolabeled with Tc and injected intravenously into the tail of mice. The labeled antibodies, expressed as %ID / g, 99m Tc-C5V L The resulting biodistribution profile of the sdAb is presented in Figure 9. The data obtained showed that tumor uptake was approximately 1.5% ID / g at 15 min, decreasing to 1% 3 h after injection. Furthermore, with the exception of liver and spleen, the biodistribution data showed low levels of activity along with rapid clearance from blood and major organs. These results support the conclusion that the C5 V expression profile in CLBL-1 xenograft tumors is consistent with that in CLBL-1 xenograft tumors. L This was also confirmed by Western blot analysis confirming the presence of the sdAb (Figure 9).

[0052] 7. Bioconjugation of VL-DAB-SN38 To develop ADCs by utilizing the free cysteine ​​at position 80, C5 V LThe predicted three-dimensional structure of the sdAb was determined. The resulting structural model showed a classical immunoglobulin domain fold consisting of eight antiparallel β-strands arranged in two β-sheets, connected by a single disulfide bond formed between Cys23 and Cys90, forming a β-sandwich (Figure 10). Three CDR motifs are readily identifiable, with CDR3 having the larger area of ​​exposed surface. Importantly, the structure shows that the CDR1 is the most abundant CDR in the sdAb domain. L The figure shows the presence of a third cysteine ​​(Cys80) on its surface, along with a free sulfhydryl group that would normally be involved in a rabbit-specific interdomain disulfide bond with another cysteine ​​on the domain. As expected, by isolating the VL domain, Cys80 becomes exposed on the protein surface, freeing its sulfhydryl group for use in cysteine-based conjugation strategies. Therefore, the single free cysteine ​​of the C5 sdAb was modified with the cytotoxic drug SN-38 and DAB-SN-38, a molecule containing a maleimide group, connected by a ROS-responsive diazaborine linker (Figure 10). C5 V L The sdAb was successfully converted into the homogeneous targeted drug conjugate C5-DAB-SN-38.

[0053] 8. Evaluation of cytotoxic activity on cNHL cells After conjugation of VL-DAB-SN-38, its in vitro activity was evaluated. To this end, cell viability assays on CLBL-1 and Jurkat cells were performed using WST-1 reagent. As shown in Figure 11A-c, the ADC showed a dose-dependent toxic effect on cNHL cell proliferation. In contrast, VL-DAB-SN38 had no effect on the proliferation of Jurkat cells, a leukemic T cell line, reinforcing the specificity of the constructed ADC. Furthermore, cell viability in the presence of VL was also evaluated, leading to the conclusion that the antibody alone did not affect cell proliferation in both cell lines.

[0054] 9. Assessment of TopoI activity To determine the effect of VL-DAB-SN-38 treatment on DNA TopoI, we evaluated its activity. DNA TopoI activity is inhibited in the presence of SN-38, which inhibits the conversion of supercoiled DNA to relaxed DNA form. As shown in Figure 11D, the presence of higher concentrations of VL-DAB-SN-38 inhibits DNA TopoI, as we can see by the presence of supercoiled DNA form. Furthermore, VL-DAB-SN-38 appears to inhibit the enzyme in a dose-dependent manner. On the other hand, the VL portion cannot change the enzyme activity, reinforcing the effect of SN-38 present on ADC as the cause of cell death. These findings support that cell death was prompted by DNA TopoI inhibition driven by SN-38 present on ADC.

[0055] conclusion The addition of rituximab, a mAb targeting the CD20 receptor, has led to a paradigm shift in the treatment of hematological malignancies in both the first-line and refractory / relapsed settings. However, the resulting experience with alternative unconjugated mAbs targeting other tumor cell receptors has demonstrated that the clinical efficacy obtained with these mAbs is often limited.

[0056] The development of ADCs has represented the most promising strategy for optimizing the efficacy of mAb-based therapies, exploiting the selectivity of antibody-antigen binding to deliver potent cytotoxic molecules directly and specifically to cancer cells.

[0057] This emerging class of therapeutic agents has demonstrated clinical efficacy in many types of blood cancers.

[0058] Given the high cytotoxic potency of the payloads used in ADCs, even lower levels of systemic exposure may result in significant toxicity. Within this context, the present invention provides LThe aim is to develop a new generation of highly selective and specific ADC for cancer therapy involving conjugation of rabbit-derived sdAb and payload on the free exposed cysteine ​​at position 80 of the framework.

[0059] Demonstrated herein is the improved cytotoxic activity of ADCs in cancer models, confirming the potential of these molecules.

[0060] The drug delivery system disclosed herein is based on rabbit-derived antibodies for canine lymphoma as an animal model of human NHL, which has proven to be a realistic opportunity for rapid and clinically relevant translation of novel immunotherapies.

[0061] NHL is one of the most common types of cancer and one of the fastest growing cancers in humans.

[0062] Due to its striking similarity to its human counterpart, the canine lymphoma model has been proposed as a powerful framework for the rapid and clinically relevant translation of novel immunotherapies. Thus, a novel class of rabbit-derived sdAb-based ADC for the treatment of cNHL that serves as an animal model for hNHL is disclosed herein.

[0063] Due to their unique B cell ontogeny, rabbit antibody-derived libraries exhibit a highly unique and diverse antibody repertoire, enriched in in vivo excised binders of high diversity, specificity and affinity. Importantly, because rabbits are evolutionarily distant from mice and rats, epitopes that are not immunogenic in rodents can increase the epitopes that can be recognized and targeted by rabbit mAbs and facilitate the generation of mAbs that cross-react with other species, an important aspect for clinical translation. Immunization of rabbits with intact B cell canine lymphoma primary cells generated specific and selective high titer antisera against NHL epitopes in all animals.

[0064] The strong and specific responses generated allowed the construction of a highly diverse and representative antibody library. The best sdAbs targeting the antigen in its natural environment were then selected using a strategy of in vitro whole-cell phage display followed by in vivo phage display in an NHL xenograft model.

[0065] This method allowed us to select phages that both bind to the tumor surface and internalize. One of the peculiarities of this final in vivo selection is the naturally occurring negative selection, which allows the reduction of off-target tissue and protein interactions by removing non-specific ligands and enriching the recovery of target-specific ligands.

[0066] To our knowledge, this is the first time that in vivo selection has been applied to the selection of sdAbs against lymphoma malignancies. Overall, the data presented here reinforce in vivo phage display selection as a powerful technique with the ability to expand the repertoire of tumor receptors that can be targeted while simultaneously confirming epitope availability in vivo and generating new antibodies for the target.

[0067] In this regard, ELISA screening allowed the selection of the best sdAb candidates targeting cNHL in terms of binding activity and expression. At the same time, NGS analysis was performed to compare the recovered in vivo biopanning with the library and selected clones. NGS analysis allowed us to show the specificity obtained by phage display compared to the initial library. Furthermore, once the most common sequence on biopanning was identified as one of the best six clones selected by ELISA, we were able to validate the ELISA screening performed via NGS.

[0068] Based on its binding and expression characteristics, C5I was shown to be the most promising sdAb targeting NHL and was selected for further characterization by FACS and immunofluorescence. Characterization studies allowed us to verify the interaction of C5 with CLBL-1 and subsequent internalization. The specificity of the antibody-antigen interaction and subsequent internalization of the complex are essential for the success of ADCs and the mitigation of off-target effects. Thus, internalization combined with the unique features of sdAbs, such as their reduced size, makes them great candidates for binding to other molecules without affecting their activity or stability.

[0069] The selection of a potent payload is important to optimize the already proven benefits of our antibodies. SN-38 is the active metabolite of irinotecan, derived from camptothecin. This molecule interacts with topoisomerase I (TopoI), which plays a fundamental role during transcription and replication. SN-38 acts as a TopoI inhibitor by binding and stabilizing the TopoI-DNA cleavage complex, resulting in DNA damage and then apoptosis when transcription and replication occur. There is evidence that the sensitivity of cells to topoisomerase poisons depends on the amount of enzyme inside the cell. Cancer cells express higher yields of TopoI, resulting in 14-16 times higher expression than in normal cells. This increased yield of the enzyme is particularly observed in certain types of cancer, including NHL.

[0070] Considering the potential of this payload, we bioconjugated C5I with SN-38 using a DAB linker to generate the novel ADC-VL-DAB-SN-38.The obtained data showed that VL-DAB-SN38 promoted cell death on canine lymphoma cells.

[0071] Furthermore, the results indicated that the cytotoxicity of VL-DAB-SN-38 against canine lymphoma was associated with DNA Topo I inhibition. Notably, these results demonstrated that the generated ADCs were stable and exhibited high cytotoxic activity against canine diffuse large B-cell lymphoma in the nM range, highlighting the potential of these rabbit-derived sdAbs as ADC moieties.

[0072] II. Description of the method for producing BBB single domain antibodies according to a second preferred embodiment of the invention As mentioned above, the present invention also relates to the development of a drug delivery system comprising a single domain antibody (sdAb).

[0073] Thus, in a second aspect of the present invention, drug delivery systems are developed to target BBB endothelial cell receptors in the central nervous system (CNS). These systems comprise rabbit-derived single domain antibodies (sdAbs) conjugated to the surface of liposomes that encapsulate appropriate drugs to enable efficient blood-brain barrier (BBB) ​​translocation. The respective production methods are also disclosed herein.

[0074] To this end, the construction of a rabbit-derived single domain antibody (sdAb) library against BBB endothelial cell receptors is disclosed herein. The sdAb antibody library can be used in in vivo phage display screening as a functional selection of novel BBB-targeting antibodies. After three rounds of selection, next-generation sequencing analysis, in vitro brain-endothelial barrier (BEB) model screening and in vivo biodistribution testing, five potential sdAbs were identified, three of which reached more than 0.6% ID / g in the brain.

[0075] To verify the proof-of-concept of brain drug delivery, the most promising sdAb, i.e., RG3 (SEQ ID NO: 44), was conjugated to the surface of liposomes encapsulating a model drug, pan-histone deacetylase inhibitor (PAN). The translocation efficiency and activity of the conjugated liposomes were determined in a dual-functional in vitro BEB-glioblastoma model. RG3-conjugated PAN liposomes enable efficient BEB translocation and exhibit potent antitumor activity against LN229 glioblastoma cells without affecting the integrity of BEB.

[0076] 3. Results and Discussion 3.1. Construction of immunized sdAb library and validation of antibodies To address the complexity of the BBB, we developed a phenotypic antibody search and identification approach that takes into account the native conformation of BBB cell surface receptors (Figure 12).

[0077] A nanobody sdAb library was constructed by whole-cell immunization of two New Zealand White rabbits with a cell line (bEnd.3) of mouse brain endothelial cells. Rabbit antibodies are well known for their ability to produce high-affinity and site-specific antibodies, which can generate antibodies that recognize similar epitopes from different species. More importantly, in contrast to other rodents, rabbits generate highly diverse and potent immune responses, especially against low-abundance proteins or hidden epitopes that are common in the BBB receptorome.

[0078] The rational use of the bEnd.3 cell line will be linked to subsequent in vivo assays for validation of our immunization strategy.

[0079] To monitor the immunological immune response of rabbits, blood was collected before and after each boost and assessed by ELISA (FIG. 13A) and Western blot (WB) (FIG. 13B) to confirm serum specificity and titer against bEnd.3 surface receptor. Results obtained for sera from two rabbits showed that the immunization method generated a strong and specific immune response against the BEB cell model receptor and there was no antibody recognition of the bEnd.3 receptor in the pre-immune serum.

[0080] Considering that the primary goal of the present invention is the identification of antibodies capable of translocating the BBB, the ability of the final serum from each rabbit to cross the BBB is evaluated in a well-characterized in vitro BEB model consisting of a monolayer of brain endothelial cells.

[0081] To determine the translocation properties of rabbit polyclonal serum, purified final serum was added apically and incubated for two time points selected based on previous optimization assays. As shown in Figure 14A, WB analysis of rabbit final serum confirmed the presence of antibodies capable of translocating to the basal compartment of the monoculture barrier model, with translocation observed at 15 and 60 min.

[0082] All in vitro BEB model assays were monitored for cell integrity after antibody incubation based on barrier permeability to FD40 probe translocation. Paracellular leakage was negligible in all cases.

[0083] Despite its high reproducibility, a major weakness of the in vitro BEB model is the reduced complexity of receptor expression compared to in vivo, which may bias the antibody validation process. Therefore, to confirm that rabbit-derived antibodies were reaching the brain in a more dynamic model, purified rabbit serum was injected intravenously into CD1 mice (and 2 and 60 min post-injection (pi)) and recovered from the blood and brain by immunoprecipitation with protein A. As shown in Figure 14B, antibodies from both immunized rabbits were detected in the mouse brain, validating the immunization strategy and the presence of antibodies with BBB-crossing properties.

[0084] 3.2. In vivo phage display selection of BBB-targeting sdAbs After immunization of rabbits and verification of the immune response to brain endothelial cells, the selection of the best antibody for BBB translocation was performed. For this purpose, the antibody light chain variable region (V) was extracted from the bone marrow and spleen cDNA of two immunized rabbits. L The sdAb library was constructed by amplifying V L The sdAb was cloned into the pComb3X phagemid vector and 1.2×10 8 The resulting phage display library represents a diversity of .

[0085] The main advantage of using sdAbs is the possibility of generating large libraries that can be used to screen a diverse set of receptors, allowing the exploration of new targets. A powerful technique for antibody selection is phage display, since antibodies displayed on the phage surface can be selected in the complex environment of animals based on desirable pharmacokinetics and target specificity. Furthermore, screening of highly specific BBB-crossing antibodies should preferably be performed in vivo, since the antibodies must be functionally identified and tested, overcoming natural barriers and mechanisms of degradation.

[0086] Thus, in vivo phage display selection was performed to select a panel of rabbit-derived brain-targeting sdAbs in the native context that retained in vivo BBB properties and their innate intracellular interactions with surrounding cells and intraluminal blood flow (Figure 15A). Briefly, the phage display library (input) was injected into the tail vein of CD1 mice. At 2 or 60 min pi, mice were perfused, euthanized, brains were removed, phages were harvested and brain-specific sdAbs were harvested. Time points were chosen based on preliminary assays with naive libraries (data not shown) to aid in the selection of sdAbs that could rapidly target the BBB, but had limited brain accumulation properties. Two subsequent rounds of selective screening were performed to enrich the BBB-targeting sdAb population (Figure 15A). At the end of the third round, recovered phages exhibited a titer of 105 (phage / mL), reflecting a high enrichment of phages with an increased ability to reach the BBB.

[0087] In contrast, no enrichment was observed when M13 helper phage was used as a control (Figure 15A). Thus, the implemented in vivo phage display approach allows the selection of sdAbs in a high stringency and restrictive environment that allows the simultaneous subtractive selection of non-specific sdAbs.

[0088] 3.3. Screening for antibodies directed against the BBB To characterize the diversity of the enriched sdAb population and identify dominant clones, individual colonies recovered from the second and third rounds of in vivo selection were randomly picked and analyzed by Sanger sequencing. Bioinformatics analysis of 64 sequenced clones (SEQ ID NO:44 to SEQ ID NO:108) showed the presence of 27 distinct groups, 6 of which contained 4 or 5 or more representatives.

[0089] The most common sequences are repeatedly selected, but there are limitations in terms of the simultaneous sequencing process, and Sanger sequencing is limited to 10 5This gives a broader overview of the phage display library of the phage display vector. To gain further insight into the enriched sequences, next-generation sequencing (NGS) of the third biopanning repertoire (Figure 4B) was performed. NGS allows large-scale sequence analysis of the panning population, allowing a genomic assessment of the diversity of the library and the frequency of each clone.

[0090] After NGS analysis and subsequent sequence filtering, we obtained 65,701 sequences from the 2 min selection panning and 35,472 sequences from the 60 min selection panning. L This decrease in fragment diversity may be due to the increased stringency of the selection method: the percentage of singletons (sequences represented by only one count) was similar in both libraries, i.e., 42.2% for the 2 min library and 46.3% for the 60 min library of all sequences.

[0091] Sequence comparison of the main clones recovered from each biopanning showed that the lead sequences were identical in terms of prevalence at both time points, but showed a higher prevalence in the 60 min repertoire (N=5336, representing 15.0% of the total sequences) compared to the 2 min repertoire (N=4548, representing 6.9% of the total sequences) (Figure 15B). Interestingly, among the top sequences derived from each time point, higher sequence conservation was observed in the complementarity determining regions (CDRs), with most differences mainly assigned to the antibody family level and framework (FR) regions.

[0092] After sequence analysis, representative most common clones were picked and characterized for antibody expression and solubility. The most stable and predominant clones, RG3, RG7, RG15, RG22 and RG23 (SEQ ID NOs: 44-48, respectively), were selected for further expression, purification and characterization for BEB translocation. The ability of individual clones to cross the BBB was first evaluated in an in vitro BEB model. This model allowed us to understand the behavior, stability and permeability of each selected sdAb in the cellular environment (Figure 16A). Briefly, each clone was added to the apical chamber and incubated for 90 min. At the end of each assay, the apical and basal volumes were collected and analyzed by WB. In this study, a direct comparison of selected BBB-targeting sdAbs was performed with VHH FC5, a promising camelid antibody selected from a non-immunized phage library targeting TMEM30A. As shown in FIG. 16B, all selected clones were able to migrate through the bEnd.3 cell monolayer, with clones RG3, RG22 and RG23 showing increased BEB crossing efficiency in vitro. This assay allowed us to confirm the selection of highly competent brain targeting and BBB crossing sdAbs. A key characteristic of a potential antibody-based therapy is clearly its targeting ability. This is even more important for CNS approaches due to the inherent selectivity of the BBB. To prove the BBB targeting and translocation ability of the selected antibodies and to determine the brain accumulation of the best clones, 99m Tc(CO) 3 Labeled individual sdAbs were injected intravenously into the tail vein of CD1 mice and ex vivo radioactivity was measured in individual organs. Biodistribution results of the tested clones at 2 and 60 min pi are presented in Table 1 and expressed as percentage of injected activity per gram of organ (%IA / g±SD). Excretion of total radioactivity is also presented as percentage of total injected activity (%IA). For comparison, Table 1 also shows the results of the control antibody, 99m Tc(CO) 3 Also included is the biodistribution profile of labeled FC5.

[0093] Table 1 shows 99m Tc(CO) 3 The biodistribution profile of the labeled sdAbs is presented. To verify that the selected clones from the phage display selection were the most competent sdAbs for BBB translocation, an in vivo biodistribution assay was performed. The sdAbs were 99m Tc(CO) 3 (H 2 O) 3 The antibody was radiolabeled with 100 mM KOH and injected intravenously into the tail vein of CD1 mice. Mice were sacrificed by cervical dislocation at 2 and 60 min pi, and the radioactivity in each organ was measured with a dose calibrator. Uptake in the brain and tissues of interest was calculated and expressed as a percentage of injected activity per gram of tissue (%IA / g).

[0094] [Table 1]

[0095] Statistical analysis of the results showed that at 2 min pi, there were no significant differences between the clones under evaluation and FC5 in many organs and tissues (blood, intestine, spleen, heart, muscle, bone and stomach), reflecting the short time point after administration. However, significant differences were observed in the uptake of organs related to the excretion route (liver and kidney) as well as in the lung and brain. In fact, all tested clones exhibited brain accumulation above 0.4% IA / g at 2 min pi, with clones RG3 and RG15 showing the highest brain accumulation, reaching values ​​of 0.82 and 0.61% IA / g, respectively.

[0096] Both radiolabeled sdAbs accumulated primarily in the kidney, which may be related to their preferential renal excretion pathway and tubular reabsorption. At 60 min pi, significant differences between clones and FC5 were observed in radioactivity uptake in all evaluated organs and tissues except brain. Significant differences were also observed in the rate of total excretion.

[0097] Regarding brain uptake, biodistribution data confirmed a lower accumulation at 60 min pi, indicating that the sdAbs translocate the BBB and are rapidly recycled back into the bloodstream and cleared from the animal's body, possibly indicating the presence of receptors on the luminal and basolateral sides of endothelial cells. This non-retention effect strengthens the potential of the selected sdAbs as drug delivery vectors that can circumvent the brain accumulation problem and its induced toxic effects. Among the selected clones for in vivo testing, 99m Tc(CO) 3 4 / 5 of the labeled sdAbs exhibited similar or superior brain accumulation compared to FC5. Among the panel of selected sdAbs, superior brain accumulation was observed for the RG3 clone (0.82±0.05% IA / g), placing it, to the best of our knowledge, as one of the most qualified sdAbs in BBB translocation described so far.

[0098] Validation of RG3 as a BBB drug delivery vector A major goal of our study was also to exploit the potential of our selection platform to identify nanobodies for targeted drug delivery approaches. To achieve this, the most qualified sdAb, RG3, along with the control FC5, was modified on the surface of liposomes encapsulating the HDACi PAN and their BBB translocation properties and antitumor activity were verified in a dual-functional in vitro BEB-glioblastoma model. Glioblastoma is an aggressive brain tumor that is highly resistant to chemotherapy and has very limited therapeutic strategies due to poor drug penetration through the BBB. Among the promising strategies for cancer treatment, PAN has emerged as a new class of highly efficient anticancer drug. Nevertheless, PAN, like many other chemotherapeutic agents, does not cross the BBB.

[0099] Among a wide panel of nanoparticles, liposomes are at the forefront of nanocarrier-based strategies for glioma therapy. Liposomes are lipid vesicles composed of one or more concentric lipid bilayers separated by aqueous compartments. Due to their unique characteristics, incorporation of both hydrophobic and hydrophilic compounds, along with their biocompatibility, long circulation time, sustained drug delivery and ability to be conjugated with targeting moieties, liposomes have remarkable potential as brain-targeted carrier systems. Moreover, nine FDA-approved liposome-based formulations for cancer therapy further support the therapeutic potential of this lipid-based carrier. Thus, PAN-encapsulated liposomes conjugated with our BBB-sdAb can be used as a novel targeted drug delivery system.

[0100] Thus, PAN-loaded liposomes with an average size of 110 nm and an encapsulation efficiency of 65±2% were successfully developed using the lipid composition DPPC:Chol:DSPE-PEG:DSPE-PEG-Biotin with a molar ratio of 1.85:1:0.14:0.01. No significant differences in encapsulation efficiency and vesicle size were observed between biotinylated and non-biotinylated liposomes.

[0101] To evaluate the cytotoxicity of unconjugated and BBB-sdAb-conjugated PAN-loaded liposomes against glioblastoma, cell viability assays were performed using the glioblastoma cell line LN229. Similar to the free PAN formulation, PAN-loaded liposomes showed potent activity and dose-dependent inhibitory effects on the proliferation of LN229 cells. Furthermore, antibody conjugation on the liposome surface did not affect the inhibitory effect of PAN-loaded liposomes on the proliferation of LN229 cells, and no significant difference was observed between liposomes conjugated with FC5 or RG3. In contrast, no cytotoxic activity was observed for RG3 and FC5 liposomal formulations without PAN (Lip-RG3 and Lip-FC5). Concurrently, the translocation efficiency of Lip-RG3 and Lip-FC5 in an in vitro single cell model was evaluated. Here, rhodamine-loaded liposomes functionalized with RG3 and FC5 were added to the apical side of the model and collected after 90 min, 6 and 24 h of incubation, and the RG3-liposomes reached a translocation maximum at the bottom of the model of 29.3 ± 6.5% at 24 h, 9.4-fold higher than the non-conjugated liposome formulation. The percentage of translocation for FC5-conjugated liposomes was then 12.9 ± 1.6%, only 4.2-fold higher than the non-conjugated liposome formulation (Figure 17). Furthermore, monitoring of BEB integrity showed that both liposome formulations had no significant effect on the barrier structure.

[0102] Finally, the antiproliferative activity of PAN loaded in RG3 and FC5 conjugated liposomes after BBB translocation was determined in a dual-functional in vitro BEB-glioblastoma model (BEB-GBM) (Figure 18). To develop the BEB-GBM model, a non-contact co-culture system was implemented consisting of bEnd.3 as the BBB barrier and LN229 cell line at the base as the GBM cell line. To evaluate the cytotoxicity against bEnd.3 cells and determine the most suitable PAN concentration to be tested in the BEB-GBM model, increasing concentrations of free PAN were added to the apex of the BEB-GBM model and their effect on BEB integrity and LN229 cytotoxicity was determined. As shown in Figure 18A, no significant effect on BEB integrity and LN229 viability was observed when no PAN was added below 2.5 μM. Thus, the BEB translocation efficiency and drug delivery of PAN encapsulated in RG3- and FC5-functionalized liposomes were evaluated by adding the liposomes to the apex of the BEB-GBM model after 24 h of incubation at various concentrations (0.1-2.0 μM). As shown in Figure 18B1 and B2, both RG3- and FC5-functionalized PAN liposomes were able to translocate in the in vitro BEB-GBM model and showed a dose-dependent inhibitory effect in the LN229 cell line without affecting the overall barrier integrity of the single-cell model.

[0103] To confirm that the cytotoxic effect of RG3 and FC5 conjugated liposomes on GBM cell lines was associated with histone acetylation induction, the H3 acetylation status of cells treated with PAN loaded in RG3 and FC5 conjugated liposomes, a key molecular mechanism of HDACi, was compared with that of unloaded liposome formulations and vehicle / control treated cells. Immunoblotting analysis showed that LN229 GBM cell lines exhibited a high acetylation status 24 hours after treatment with both RG3 and FC5 PAN loaded liposomes when compared with unloaded liposomes and vehicle / control treated cells. A final in vivo proof-of-concept biodistribution study was performed to demonstrate the BBB translocation of our RG3 functionalized liposome system. To this end, 111 RG3-functionalized liposomes radiolabeled with indium were prepared and administered to CD1 mice.

[0104] As shown in Table 2, 2 min after intravenous injection (iv), 1% of the injected dose per gram of tissue (%ID / g) of our developed radiolabeled system reached the brain, and this amount remained constant for 60 min after administration. In contrast, significantly lower brain uptake was observed for non-conjugated liposomes. Statistical analysis of biodistribution and excretion data at 1 and 24 h pi between RG3-conjugated liposomes and control liposomes showed significant differences in almost all tested organs and tissues, except for the intestine and stomach, at 1 h pi. Indeed, significantly higher activity was observed in the bloodstream, and higher uptake was observed in many organs. The rate of excretion of total radioactivity for RG3-conjugated liposomes was significantly lower (15.4±2.5% and 28.2±2.7%IA at 1 and 24 h, respectively, versus 59.7±6.6% and 68.3±0.4% for control liposomes). At 24 h pi, significant differences remained for the major organs, i.e., blood, brain, excretory organs (kidney, liver, intestine), as well as total radioactivity excretion.

[0105] At the same time, in vivo and in vitro data showed that our selected BBB-sdAb and developed sdAb-liposome system have high ability to cross the BBB, which is an advantageous strategy for CNS targeted therapy. Table 2 presents the biodistribution of selected RG3-conjugated liposomes. To verify the BBB translocation of RG3-conjugated liposomes, in vivo biodistribution assay was performed. RG3-conjugated liposomes were radiolabeled with 111In and intravenously injected into the tail vein of CD1 mice. Mice were sacrificed by cervical dislocation at 2 min, 60 min and 24 h after injection, and the radioactivity of each organ was measured using a dose calibrator. The uptake in brain and desired tissues was calculated and expressed as a percentage of the injected radioactivity per gram of tissue (%ID / g). The excretion of total radioactivity was expressed as a percentage of the injected activity (%IA).

[0106] [Table 2]

[0107] conclusion The present invention shows that using an immunized rabbit-derived sdAb library developed against BBB epitopes and an in vivo phage display selection method, a panel of nanobodies was identified that exhibited one of the highest levels of BBB translocation ever described. Furthermore, the developed RG3-liposomes specifically target and translocate the BBB, delivering payloads at effective concentrations in an in vitro setting, and are strong candidates for drug delivery to the CNS. Thus, the proposed in vivo sdAb development platform is a pioneering selection method for highly specific nanobodies with promising properties for brain targeting and drug delivery to various CNS diseases, such as brain tumors, Alzheimer's disease, or Parkinson's disease. [Example] EXAMPLES

[0108] Canine multicentric lymphoma biobank Patients with canine multicentric lymphoma were followed in the Oncology Unit of the Teaching Hospital of the Veterinary Medicine Faculty - University of Lisbon (FMV / UL), where clinical evaluation was performed. At a preliminary stage, a complete medical history, clinical signs and physical examination were evaluated for diagnosis and staging. Complete blood count and biochemistry profile, as well as abdominal and thoracic imaging, were performed. Histopathological evaluation of lymph nodes was performed after lymph node biopsy. This histopathological evaluation included morphological examination, classification of lymphoma into grade subcategories and immunophenotyping to determine the immunophenotype present - B or T. Immunohistochemical markers included CD3, CD20, CD79αcy and PAX-5. This clinical and laboratory examination allowed the dogs to be staged using the World Health Organization (WHO) system.

[0109] Inclusion criteria included dogs with recently diagnosed multicentric lymphoma by clinical examination and cytology of lymph node fine needle aspiration that had not yet started therapy. Exclusion criteria included dogs that had started chemotherapy within the last 8 weeks of study enrollment, received steroids or other immunotherapeutic agents, or had severe disease.

[0110] All sample collections were conducted in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Animals, with written consent from the owners, and approved by the Animal Care and Use Committee of FMV / UL.

[0111] Blood samples allowed the isolation of plasma and serum and the extraction of DNA (Dneasy Blood & Tissue, Qiagen, Hilden, Germany) and mRNA (Rneasy Protect Animal Blood System, Qiagen), which were stored at -80°C.

[0112] Furthermore, PBMCs were isolated by the Ficoll gradient method (Biocoll Separating Solution, BioChrom®, Fisher Scientific, New Hampshire, USA), and after cell viability evaluation, 5 × 10 6 Aliquots of cells were suspended in 90% fetal bovine serum (FBS) (Gibco, Life Technologies, Paisley, UK) and 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Missouri, USA) and kept in liquid nitrogen.

[0113] Sterile lymph node biopsies were divided and after lymphoma cell isolation, one third was finely cut and stored in RNAlater® (Invitrogen, Life Technologies, Paisley, UK) at -80°C, one third was formalin fixed and one third was stored in liquid nitrogen. Briefly, solid tissues were cut, passed through a cell strainer (Cell Strainer, BD Falcon®), suspended in Roswell Park Memorial Institute-1640 (RPMI-1640) medium (Gibco) supplemented with 20% FBS and 100 U / ml penicillin and 0.1 mg / ml streptomycin (Gibco), and isolated by Ficoll gradient (Biocoll Separating Solution, BioChrom®).

[0114] For assessing cell viability and storage, 5 x 10 6 Aliquots of cells were suspended in 90% FBS and 10% DMSO and kept in liquid nitrogen. Clinical follow-up information for all cases was collected from electronic medical records. All dogs included in this study were client-owned animals and entered the study during their diagnostic evaluation. All sampled animals stayed with their owners after sample collection. EXAMPLES

[0115] Cell lines and culture The canine B-cell lymphoma cell line CLBL-1 was kindly provided by Dr. Barbara Rutgen (University of Vienna, Austria). Human Burkitt's lymphoma Raji cell line, human T lymphocyte cells and human cell line HEK293T cell line (suitable for ectopic expression of mammalian proteins) were obtained from the American Type Culture Collection (ATCC, American Type Culture Collection, Manassas, VA). CLBL-1, Raji and Jurkat cell lines were maintained in RPMI-1640 medium (Gibco) supplemented with 10% FCS (Gibco) and 100 U / ml penicillin / 0.1 mg / ml streptomycin (Gibco). HEK293T cell line was cultured in DMEM medium supplemented with 10% FCS (Gibco) and 100 U / ml penicillin / 0.1 mg / ml streptomycin (Gibco). All cell line cultures were incubated at 37 °C in 5% CO 2 The cells were maintained at 37°C in a humidified atmosphere (T75 tissue culture flasks, Greiner Bio-One, Kremsmunster, Austria). EXAMPLES

[0116] Rabbit immunization with cNHL primary cells Three female New Zealand White rabbits (Charles River) were cultured with 1 × 10 7The rabbits were immunized with cNHL primary cells and boosted over a period of 4 months to induce a strong and specific immune response against the NHL receptor. Cells were selected from patients 5 and 6 from our biobank, who were diagnosed with diffuse large B-cell lymphoma (DLBCL). For this purpose, tumor cells isolated from lymphoma-affected lymph nodes were thawed, washed in PBS, and resuspended in 1 ml of PBS after checking cell viability. Injections were administered subcutaneously at 2-week intervals. Before each immunization, blood was collected from the marginal ear vein for serum isolation. Five days after the last boost, the rabbits were sacrificed by cardiac puncture exsanguination after propofol anesthesia, and the spleen and bone marrow were harvested for total RNA isolation and cDNA synthesis. EXAMPLES

[0117] Characterization of rabbit immune responses The rabbit immune response against the biobanked cNHL primary cells and CLBL-1 cells was monitored by ELISA serology. Pre-bleed serum was used as a control. Briefly, 50 × 10 3 Cells were blocked with PBS-BSA 1% (BSA, bovine serum albumin, Merck) for 30 min, washed with PBS, and incubated with serial dilutions of rabbit serum (1 / 1000 to 1 / 32000) for 1 h. Cells were then washed with PBS, and secondary antibody goat anti-rabbit IgG-Fc specific HRP (Jackson ImmunoResearch) at 1:3000 in PBS-BSA 1% was added to each well and incubated for 1 h. After incubation, ABTS substrate solution (Merck) was added, and optical density (OD) was measured at 405 nm using a microplate reader (Bio-Rad).

[0118] Each serum was also analyzed for its binding properties to Cnhl cells by FACS. For this purpose, CLBL-1 and Cnhl cells were prepared from patients 5 and 6. The cells were washed twice in PBS-BSA 0.5% and incubated with rabbit pre-bleed and terminal bleed (1:3000) for 30 min at 4° C. The cells were then washed three times with cold PBS-BSA 0.5% and incubated with secondary antibody (Alexa Fluor® 647 goat anti-rabbit IgG antibody) at 1:10000 in PBS-BSA 0.5% for 30 min at 4° C. The cells were washed three times with cold PBS-BSA 0.5% and submitted for FACS analysis (FACSCalibur).

[0119] Unstained cells were used as negative control for voltage settings. For multicolor sorting, single-color controls were used for compensation settings. Data were analyzed by FlowJo software version 10 (FlowJo LLC).

[0120] To evaluate the protein profile recognized by the rabbit sera, immunoblotting was performed using CLBL-1 cells, Cnhl primary cells (B1 and B2) and PBMCs (C1 and C2) from healthy dogs. Peroxidase-conjugated goat anti-rabbit antibody (Jackson Immune Research) was used as the secondary antibody. EXAMPLES

[0121] Construction of a single domain antibody library Total RNA was extracted from the spleen and bone marrow of each rabbit using Trizol reagent according to the manufacturer's instructions (Invitrogen). First strand cDNA was synthesized using Transcriptor High Fidelity (Roche) according to the manufacturer's instructions. The first strand cDNA from each rabbit was then subjected to separate 30 cycles of polymerase chain reaction using Phusion High Fidelity DNA polymerase (Thermo Fisher Scientific) and a combination of 10 specific oligonucleotide primers for the amplification of rabbit sdAbs in the previously described light chain variable region (VL) (9xVkappa and 1xVlambda) format. The PCR products encoding the library of antibody fragments (sdAbs) were then gel purified, restriction digested with SfiI, and cloned into Pcomb3Xss. The ligated products were then transformed into electrocompetent cells by electroporation, and the library was titrated. To confirm the insertion efficiency and diversity of the library, PCR colony was performed using primers RSC-F and RSC-B. Phage library sequencing was performed by GATC Biotech AG (Ebersberg, Germany) using pComb3x ATG primers. Vector NTI Advance 10 software (Thermo Fisher Scientific) was used to translate amino acid sequences and evaluate homology. EXAMPLES

[0122] Phage display selection of antibodies targeting cNHL V LThe phage library displaying sdAbs was first panned using a subtractive cellular phage display protocol previously described by Carlos Barbas and our work (Barbas III, CF, Burton, DR, Scott, JK & Silverman, GJ Phage Display: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, 2001) and Dias, JNR et al. Characterization of the canine CD20 as a therapeutic target for comparative passive immunotherapy. Sci Rep 12, 2678 (2022)), which included negative selection on HEK293T cells followed by positive selection on CLBL-1 cells. Then, after three rounds of in vitro selection, further panning was performed in vivo in a xenograft CLBL-1 mouse model. Briefly, female 6-8 week old SOPF / SHO SCID mice (Charles River) were maintained in microisolation cages under pathogen-free conditions. Mice were allowed to acclimate for at least 2 weeks before the start of the experiment. They were then cultured in PBS containing Matrigel (Corning, NY, USA) at 1 × 10 6 Tumors were induced by subcutaneous injection of CLBL-1 cells (1:1) into the dorsal interscapular region. 3 When a minimum volume of 100 μl of phage (1 × 10) was reached, 100 μl of freshly prepared phage (1 × 10) from the third in vitro selection round was injected into the tail vein of three SCID mice. 10pfu / ml) were injected intravenously. After 60 min of phage circulation, mice were sacrificed, perfused, and xenograft tumors were removed and weighed. After tumor homogenization in a 70 μm cell strainer (VWR, Radnor, PA, USA), phages were harvested by incubating homogenized tumors with 500 μL of freshly prepared trypsin (1 mg / ml) (Gibco) supplemented with anti-protease antibody (Merck) and DNAse (1 U / μL) (Invitrogen) for 15 min at 37°C. Eluted phages (binders) were then harvested after centrifugation at 10,000×g for 10 min at 4°C and normalized to a final volume of 1 mL in PBS. To elute internalized phages, the cell pellet obtained after trypsin elution was washed three times with PBS and centrifuged at 10,000×g for 5 min at 4°C. The cell pellet was then resuspended with 200 μl of 0.1 M triethylamine, incubated for 10 min, and neutralized with 50 μl of 1 M Tris 7.5. The eluted phages were normalized to a final volume of 1 ml. The resulting output phages (binders and internalization factors) were used for phage titration and reamplification in E. coli ER2738 (Lucigen) cells for storage and lead selection. Phages were also titrated in blood. Unrelated naïve rabbit V L The sdAb library and M13 helper phage were used as controls in pilot studies. EXAMPLES

[0123] ELISA screening of antibodies targeting cNHL To express and select anti-NHL-sdAbs, phagemid DNA encoding selected anti-cNHL sdAbs was cloned into the PT7-PL (PT7-peptide leader) vector and transformed into E. coli strain BL21. Individual colonies were inoculated into 100 μl Superbroth (SB) medium containing the Overnight Express™ Autoinduction System (Novagen®) and 100 μg / ml ampicillin and incubated overnight at 30° C. The next day, 40 μl BugBuster (Roche) containing anti-protease cocktail-EDTA free inhibitors (Roche) was added and incubated for 30 min at 4° C. Plates were then centrifuged at 1200 rpm and supernatants were tested in an ELISA assay. Three different conditions were assessed: binding of sdAbs to antigen, expression levels and non-specific binding. After coating the wells with CLBL-1 or Raji cell extracts for 1 h at 37°C, the wells were blocked with 3% BSA in PBS. The wells were then washed with PBS, clones were added and incubated for 1 h at 37°C. The plates were then washed and incubated with anti-HA HRP antibody (Roche). Finally, after 1 h of incubation, the plates were washed, ABTS (Roche) was added and the optical density at 405 nm was measured at various time points. To evaluate the expression levels, the same protocol was applied, except for the antigen coating.

[0124] For non-specific binding, the antigen was replaced with 3% BSA. Rabbit serum and anti-CD20 antibody were used as positive controls. BL21 cell extract was used as negative control. Finally, the 10 best individual clones were sequenced at Eurofins. Sequence analysis was performed using Vector Nti software (Invitrogen). Numbering and sequences of antibody frameworks, CDRs and amino acids followed the rules described by Kabat et al. (1991) Sequences of proteins of immunological interest. Bethesda, MD, US Dept. of Health and Human Services, Public Health Service, National Institutes of Health. The obtained sequences were compared and aligned with the NGS data using the above mentioned Vector NTI software (Invitrogen). EXAMPLES

[0125] Phage display enrichment and next-generation sequencing analysis To analyze the amino acid sequence and profile of the clones selected by ELISA, 43 best individual clones were sequenced at Eurofins. Sequence analysis was performed using Vector NTI software (Invitrogen). Numbering and sequence of antibody framework, CDR and amino acids followed the rules described by Kabat et al. shown above.

[0126] Furthermore, to evaluate the diversity and enrichment achieved by phage display, we performed next-generation sequencing (NGS). To this end, we used the 250 paired-end module of the MiSeq (Ilumina) sequencing platform to sequence the initial library and the VL of the in vivo biopanning. L The complete sequence of the sdAb region was obtained. The MiSeq library for sequencing was LThe sdAb region was amplified and 2 μg of purified amplicon was then sent for sequencing at STABVIDA. Data was analyzed using Geneious software.

[0127] The resulting data was assembled by mixing paired-end sequence reads, translating the sequences into proteins, and removing all sequences that were less than 100 amino acids and did not have an Sfi cleavage site and a histidine tail section. A custom python script was then developed to organize and count the sequence reads. Finally, the resulting sequences were compared and aligned with the NGS data using the Vector NTI software (Invitrogen) mentioned above. The data was presented in a graph showing the pattern of sequence reads. EXAMPLES

[0128] Production and purification of sdAbs The three best sdAbs were selected according to their NHL cell binding capacity and one of these (C5I) was expressed and purified. To express and purify the C5I clone, DNA cloned into the PET21 expression vector (Sigma-Aldrich, St. Louis, MO, USA) was transformed into the non-repressible E. coli strain BL21(DE3) (Lucigen, Midddleton, WI, USA). C5I was produced by inoculating 10 μl of frozen clone into Superbroth (SB) medium containing 100 μg / ml ampicillin. The culture was grown overnight at 37°C and then diluted 1:30 in SB medium, 100 μg / ml ampicillin. When the culture reached OD600nm=0.6, expression of the clone was induced by the addition of 0.6 mM isopropyl-1-thio-β-D-galactoside (IPTG) and incubated overnight at 19°C. After expression, bacteria were harvested by centrifugation (4000 rpm, 15 min, 4°C) and resuspended in 50 ml starting buffer (50 mM HEPES, 1 M NaCl, 10 mM imidazole, 2 M urea, 5 mM CaCl2, 1 mM β-mercaptoethanol, and pH=8) supplemented with protease inhibitors (Roche). Cells were lysed by sonication and inclusion bodies were harvested by centrifugation (9000 rpm, 30 min, 4°C).

[0129] The pellet was washed (50 mM HEPES, 1 M NaCl, 10 mM imidazole, 2 M urea, 5 mM CaCl2, 1 mM β-mercaptoethanol, and pH=8), sonicated, and centrifuged (9000 rpm, 30 min, 4° C.). The inclusion bodies were then resuspended in 6 M urea buffer (50 mM HEPES, 1 M NaCl, 10 mM imidazole, 6 M urea, 5 mM CaCl2, 1 mM β-mercaptoethanol, and pH=8) and incubated overnight at 4° C. under stirring for protein denaturation. A final centrifugation step was performed to remove cell debris and the supernatant was filtered through a 0.2 μm syringe filter. The denatured sdAb was purified by nickel chelate affinity chromatography using the C-terminal His tag. Bound proteins were eluted in high-concentration imidazole buffer (50 mM HEPES, 1 M NaCl, 500 mM imidazole, 6 M urea, 5 mM CaCl2, 1 mM β-mercaptoethanol, and pH = 7.8). Gouveia et al., 2017 30 Refolding was performed by stepwise dialysis according to the procedure described above. C5I was then purified by size-exclusion chromatography (SEC) using a HiPrep 16 / 60 Sephacryl S-100 column (Sigma-Aldrich). Protein purity was analyzed by sodium dodecyl sulfate / polyacrylamide gel electrophoresis (SDS / PAGE) gels using 15% acrylamide gels under denaturing conditions. EXAMPLES

[0130] Immunofluorescence microscopy 1.5 × 10 cells on an ibidi μ-Slide 8 glass-bottom well (#80827, Ibidi, Germany). 5 Seed CLBL-1 cells and incubate at 37 °C in 5% CO. 2The cells were incubated at 37°C for 24 h in a humidified atmosphere at 4°C. 5 μM VL was then added to the cells and incubated at 37°C for 90 min. After incubation, the cells were washed twice with PBS, fixed with PFA 4% for 15 min at RT, permeabilized with 0.1% Triton X-100 for 10 min at RT, washed, blocked with 0.1% Triton X-100 and 3% BSA in PBS (blocking solution), and incubated overnight with anti-HA (Roche, 1:50) at 4°C. The following day, the cells were washed twice with PBS and incubated with anti-rat Alexa Fluor-488 (1:500) for 1 h at RT. After washing, DAPI Vectashield (Vector Labs, CA, USA) was added to the cells. Image acquisition was performed on a confocal scanning Zeiss LSM 880 microscope (Carl Zeiss, Germany) equipped with a Plan-Apochromat DIC X63 oil immersion objective (1.40 numerical aperture). A diode 405-30 laser was used to excite DAPI and an argon laser in the 488 nm line to excite Alexa Fluor-488. 1.80x zoom images were recorded at a resolution of 1024x1024 in Airyscan acquisition mode. ZEN software was used for image acquisition and Fiji software was used for image processing. EXAMPLES

[0131] C5 V L Characterization of sdAb binding and internalization C5 V against CLBL-1 cells LThe binding and cell internalization properties of the sdAb were examined by cytometry and immunofluorescence. For cytometry analysis, C5 was incubated with CLBL-1 cells for 90 min as described in the Materials and Methods section. The data shown in Figure 5A indicated that C5 specifically binds to CLBL-1 cells. Conversely, no binding interaction of C5 with Jurkat cells was observed (Figure 5B). Live / dead reagent was used to exclude dead cells, and background noise was also evaluated in controls with secondary antibodies (data not shown). To better characterize the binding of C5 to CLBL-1 cells, we further evaluated the internalization of C5 sdAb on cells using immunofluorescence assay. As shown in Figure 5C, a high density of Alexa Fluor-488-labeled C5 sdAb was observed in the perinuclear region. Conversely, there was no detectable fluorescence in either the control sample images or in Jurkat cells (Figure 5D). Thus, these data confirmed the binding of C5 to CLBL-1 cells and its internalization into the cytoplasm, a property that is essential for developing an effective ADC. EXAMPLES

[0132] Bioconjugation of VL-DAB-SN38 1 μL of H2O, 5 μL of compound DAB (4 mM in DMSO) and 1 μL of IS (20 mM in DMSO) were added to 993 μL of PBS pH 7.4 containing 10% DMSO to obtain the final solution of compound DAB-SN38 (20 μM, PBS pH 7.4, 10% DMSO). DAB (20 eq., 9 mM, DMSO) was added to a solution of PBS pH 7.4 containing VL (10 μM) and TCEP (1.5 eq., 3.5 mM) and the solution was mixed at 25° C. for 1.5 hours. The expected conjugate was evaluated after 1.5 hours by high-resolution mass spectrometry and recorded in a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific™ Q Exactive™ Plus). The final immunoconjugate VL-DAB-SN38 was detected. Mass spectra were analyzed using MagTran software. EXAMPLES

[0133] Cytotoxicity assay To determine the effect of VL-DAB-SN-38 on CLBL-1 and Jurkat cell proliferation, cell viability assays were performed using the cell proliferation reagent WST-1 reagent (Roche, Basel, Switzerland). Briefly, cells were cultured at 6 × 10 4 Wells were seeded at a density of 1000 x 1000 and subjected to increasing concentrations (from 2.5 mM to 12.5 nM) of each compound (VL, VL-DAB-SN38 and SN-38). After 48 h of treatment, cell viability was assessed using WST-1 according to the manufacturer's instructions. Absorbance at 450 nm was measured using an iMark microplate reader (Bio-Rad). Duplicate wells were used to determine each data point, and three independent experiments were performed on different days. Best-fit EC50 values ​​for each formulation were calculated using GraphPad Prism software (version 8.0, San Diego, CA, USA) using the logarithm (inhibitor) vs. response (variable slope) function. EXAMPLES

[0134] DNA TopoI activity assay TopoI activity on VL-DAB-SN38 was determined using a human topoisomerase I assay kit (Topogen, CO, USA) according to the manufacturer's instructions. Briefly, 30 μl of reaction containing VL-DAB-SN38 was incubated with 1× reaction buffer (10 mM Tris-HCl pH=7.9, 1 mM EDTA, 0.15 M NaCl, 0.1% BSA, 0.1 mM spermidine, 5% glycerol) and 10 U of TopoI for 1 h at 37° C. The previous reaction mixture was then incubated with supercoiled DNA for 1 h at 37° C. To stop the reaction, stop loading buffer (0.125% bromophenol blue, 25% glycerol, 5% sarkosyl) was added to the reaction. Samples were loaded onto a 1% agarose gel and run in 1×TAE buffer. The gel was then stained with ethidium bromide for 45 min and destained in distilled water. Relaxed DNA, SN38 and VL were used as controls. EXAMPLES

[0135] Biodistribution studies and tumor targeting Selected Vs were used to evaluate biodistribution and tumor targeting on xenograft models of NHL. L sdAb(C5) was chromatographed with radioactive precursor [ 99m Tc(CO) 3 (H 2 O) 3 ] +The radiochemical purity (RP) was checked by reversed-phase high-performance liquid chromatography (RP-HPLC) and instant thin-layer chromatography silica gel (ITLC-SG, Agilent Technologies, USA). 99m Tc(CO) 3 (H 2 O) 3 ] + The solution was added to a nitrogen-purged sealed glass vial containing a solution of His-tag with C5 to give a final concentration of 1 mg / ml. The mixture was incubated at 37°C for 45-60 min, after which ITLC-SG analysis was performed using a 5% HCl (6 M) solution in MeOH as the eluent. 99m Tc(CO 3 )-C5 RP was evaluated. 99m Tc(CO) 3 (H 2 O) 3 ] + and 99m TcO 4 ] - moves in front of the solvent (Rf=1), 99m Tc(CO) 3 -C5 remains intact (Rf=0). The radioactivity distribution on the ITLC-SG strips was evaluated using a miniGita Star scanning device (Elysia-Raytest, Germany) coupled with a Gamma BGO-V-Detector (Elysia-Raytest). 99m For purification and concentration of Tc-labeled sdAb, 3 K Amicon (Merck Millipore) was used. 99m Tc(CO) 3 -C5 was used for biodistribution study after RP determination by ITLC-SG. To this end, 100 μl of the solution was injected into the tail vein of mice. 99m Tc(CO)3 Mice were intravenously injected with -C5 and sacrificed by cervical dislocation at 15 min and 3 h after injection. Radioactivity was measured using a dose calibrator (Carpintec CRC-15W). After removal of the desired tumor and tissues, their radioactivity was measured using a γ-counter (Berthold, Germany). Uptake was expressed as a percentage of the injected active dose per gram of organ or tissue (%ID / g). EXAMPLES

[0136] Immunization of rabbits with BBB cells All animal handling procedures were performed in accordance with good practice and EU recommendations on animal welfare and were approved by the Animal Care and Ethics Committee. Animals were housed in a temperature- and humidity-controlled room with a 12-h light-12-h dark cycle. Two New Zealand white rabbits (Charles River) were immunized with mouse brain endothelial bEnd.3 cells (ATCC® CRL-2299™) and boosted on days 14, 28, 56 and 70 to induce a strong and specific immune response against the endogenous bEnd.3 receptor. Briefly, bEnd.3 cells were cultured in 2000 mL of PBS at 4°C for 30 min at 5% CO. 2 The mice were grown to confluence on T175 flasks in Dulbecco's Modified Eagle Medium (DMEM) medium (Gibco) containing high glucose and pyruvate supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco) in a humidified atmosphere at 37°C. Before each immunization procedure, blood was collected from the ear vein and 1 × 10 6 bEnd.3 cells were suspended in 0.5-1 ml of sterile phosphate-buffered saline (PBS) and injected subcutaneously into rabbits. Injections were administered at 2-3 week intervals. Five days after the last boost, rabbits were sacrificed by cardiac puncture exsanguination after propofol anesthesia, and spleens and bone marrow were harvested for total RNA isolation and cDNA synthesis. EXAMPLES

[0137] Characterization of rabbit immune responses The rabbit immune response against bEnd.3 cells was monitored by ELISA serology of blood taken before and after each boost injection. 4 bEnd.3 cells were seeded at 10 cells / well in a 96-well plate and incubated at 5% CO 2 The cells were incubated for 24 h at 37°C in a humidified environment containing 1% PBS-BSA (BSA, bovine serum albumin, Merck, Kenilworth, NJ, USA). The following day, the cells were blocked for 30 min with PBS-BSA 1% (BSA, bovine serum albumin, Merck, Kenilworth, NJ, USA), washed with PBS, and incubated for 1 h with serial dilutions of rabbit serum (1 / 500 to 1 / 32,000). The cells were then washed with PBS, and the secondary antibody goat-α anti-rabbit IgG-Fc specific HRP (Jackson ImmunoResearch, West Grove, PA, USA) at 1:3000 in PBS-BSA 1% was added to each well and incubated for 1 h. After incubation, ABTS substrate solution (Merck) was added, and the optical density (OD) was measured at 405 nm in a microplate reader (Bio-Rad, Hercules, CA, USA). Each serum was also analyzed for its binding profile to bEnd.3 protein extract by WB. Briefly, bEnd.3 total protein extracts obtained after RIPA cell lysis buffer (50 mM tris-HCl pH 7.4; 150 mM NaCl; 1% NP-40, 0.25% Na-deoxycholate) were separated by 11% SDS-PAGE and transferred to a PVDF membrane as previously described

[39] . WB was then performed with each serum at a 1 / 500 dilution in PBS-BSA 1% followed by goat-(alpha) anti-rabbit IgG-Fc specific HRP. Furthermore, the BBB translocation properties of each serum were evaluated in vitro and in vivo. To that end, each serum was purified by protein A chromatography as previously described

[40] and then its BBB crossing properties were evaluated as described in the in vitro BEB model and in vivo sections below. EXAMPLES

[0138] Construction of a single domain antibody library Total RNA was extracted from the spleen and bone marrow of each rabbit using Trizol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. First-strand cDNA was synthesized using the Transcriptor First-Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). First-strand cDNA from each rabbit was then subjected to separate 30 cycles of polymerase chain reaction using Phusion High Fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) and a combination of 10 specific oligonucleotide primers for amplification of rabbit sdAbs in VL (9xVkappa and 1xVlambda) format as previously described [41, 42]. PCR products were purified, digested with SfiI restriction enzyme (Roche) and cloned into appropriately cut phagemid vector pComb3X [42, 43]. The recombinant phagemid was introduced into competent E. coli ER2738 (Lucigen, Middleton, WI, USA) cells by electroporation, and the V L Phage displaying the sdAb library were produced as previously described

[39] and immediately used in in vivo phage display panning. EXAMPLES

[0139] In vivo phage display For the first selection round, three CD1 mice (Charles River, Wilmington, MA, USA) were injected with the immunization vector into the tail vein. L 100 μL of freshly prepared phages (approximately 1 × 10 11Phages were intravenously injected at 1000×g / ml. For optimization, phages were allowed to circulate for various time points (2 min, 60 min, 6 h or 24 h) before mice were sacrificed, perfused with PBS, and brains were extracted and weighed. After brain homogenization in a 70 μm cell strainer (VWR), the cell homogenate was centrifuged at 1500×g for 10 min at 4° C. The supernatant was then discarded and the cell pellet was resuspended in 2 ml of washing buffer (PBS-0.05% Tween 20), mixed with gentle agitation for 2 min at room temperature, and centrifuged at 1500×g for 10 min at 4° C. This washing step was repeated three times for the first selection round and five times for the following rounds. Phages were then harvested by incubating homogenized brain cells with 500 μL of freshly prepared trypsin (1 mg / ml) (Gibco, Thermo Fisher Scientific) supplemented with anti-protease (Merck) and DNAse (1 U / μL) (Invitrogen) for 15 min at 37° C. Eluted phages were then harvested after centrifugation at 14,000×g for 10 min at 4° C. and normalized to a final volume of 1 mL in PBS. The resulting respective output phages were used for phage titration and re-amplification in ER2738 for a new round of in vivo selection. Phages were also titrated in blood. Three rounds of in vivo selection were performed at 2 and 60 min after the pilot test including all the above time points. An irrelevant naive rabbit VL sdAb library and M13 helper phage were also used as controls in the pilot test. EXAMPLES

[0140] In vivo phage display enrichment and next-generation sequencing analysis To analyze the enrichment and profiles obtained after each round of in vivo phage display selection, we first LIndividual clones were randomly selected from the second and third selection rounds of the sdAb library and sequenced at Eurofins (64 clones in total). Sequence analysis was performed using Vector NTI software (Invitrogen) and antibody framework, CDR and amino acid numbering and sequence alignment were performed as defined by Kabat et al.

[44] . To further analyze the global diversity and enrichment obtained, we performed NGS. To that end, we sequenced the Vs selected in the third biopanning at both time points (2 min and 60 min) using the 250 paired-end module of the MiSeq (Illumina, San Diego, CA, USA) sequencing platform. L The complete sequence of the sdAb region was obtained. The Miseq library for DNA sequencing was L The sdAb region was prepared by amplifying, and 2 μg of purified amplicon was sent to STABVIDA for sequencing. NGS sequence data was analyzed using Geneious software (Biomatters Ltd, Auckland, NZ). The sequence data was processed by mixing paired-end sequence reads, translating the sequences to protein, and discarding all sequences that were less than 100 amino acids and did not have an SfiI cleavage site and a histidine tail section. An in-house custom python script was then developed to summarize and count the sequence reads. A bar graph was generated to represent the pattern of sequence reads. EXAMPLES

[0141] V L Expression and purification of sdAbs To express and purify the selected clones, L The genes encoding the sdAbs were transferred into the Pet21a plasmid (Novagen, Birmingham, UK) and transformed into E. coli BL21(DE3) electrocompetent cells (Invitrogen). LFresh colonies of sdAb clones were grown overnight at 37° C. in Super Broth (SB) medium containing 100 μg / ml ampicillin. A 10 ml sample of cells was used to inoculate 1 liter of SB medium containing 100 μg / ml ampicillin. OD 600nm Cells were grown at 37°C until an ATP concentration of 0.6 was reached, induced with 0.6 mM IPTG, and growth was continued for 18 h at 19°C. After induction, bacteria were harvested by centrifugation (4000 x g, 4°C, 15 min) and resuspended in 50 ml of equilibration buffer (20 mM NaHCO3) supplemented with protease inhibitors (Merck). 2 PO 4 The cells were suspended in 5% CO (pH 7.4, 500 mM NaCl, 30 mM imidazole, and pH 7.4). The cells were lysed by sonication. Centrifugation (14,000×g, 4° C., 30 min) was performed to remove cell debris, and the supernatant was filtered through a 0.2 μm syringe filter. L The sdAb was purified by immobilized metal affinity chromatography (IMAC) using an HP Histrap column and the C-terminal His of Pet21a. 6 After a washing step, the eluate was eluted with a linear imidazole gradient of 60–300 mM in the elution buffer, followed by purification with the AKTA Start system (GE Healthcare, Chicago, IL, USA) using a 500 mM glycerol (V L Elution of the sdAb was performed. The eluted fractions were pooled, desalted, and concentrated in PBS using a 3K Amicon column (Merck). LThe sdAb samples were loaded onto a HiPrep 16 / 60 Sephacryl S-100 HR gel filtration column (GE Healthcare) and pooled fractions were analyzed for protein purity by 15% SDS-PAGE followed by Coomassie blue staining and WB using HRP-conjugated anti-His antibody (Roche). Protein concentration was determined by measuring absorbance at 280 nm in a Nanodrop 2000 (Thermo Fisher Scientific). The same procedure was performed to express and purify the control antibody FC5 VHH. EXAMPLES

[0142] In vitro BEB model The in vitro BEB model was optimized based on a previous study

[45] . Briefly, bEnd.3 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin (Lonza, Basel, Switzerland) antibiotic solution. Cells were incubated at 37 °C for 24 h at 5% CO 2 The cells were cultured at 37°C in a humidified atmosphere with the medium changed every other day. The cells were allowed to adhere in a monolayer and, when confluent, were harvested from the cell culture flasks using trypsin-EDTA (Gibco) and 4 × 10 3 Cells / well were seeded into 24-well plate tissue culture inserts (Falcon, Atlanta, GA, USA) pre-coated with bovine plasma fibronectin (1 mg / ml) (Merck). To allow for the formation of tight junctions, cells were incubated for 11-14 days, with medium changed every 2 days. To assess the integrity of the in vitro BEB model before and between assay days, fluorescent probes of fluorescein isothiocyanate-dextran with MW of 4 kDa (FD4) and 40 kDa (FD40) (Merck) and a stock concentration of 25 mg / ml were incubated in transport buffer (TB) (5 mM glucose, 5 mM MgCl 2 , 10 mM HEPES, pH 7.4 and 0.05% BSA) at an OD of 0.1 493nmThe probes were then added to the top side (apex) of the transwells and incubated for 2 hours. Samples were collected from the top and bottom and the fluorescence intensity was measured in a microtiter plate reader (BMG Labtech, Fluostar OPTIMA, Ortenberg, Germany) with excitation at 485 nm and emission maximum at 520 nm. EXAMPLES

[0143] In vitro BEB translocation Selected V L To determine the in vitro BEB translocation efficiency of sdAbs, 15 μg of each purified antibody was added to the top of the transwell and incubated for 15 and 90 min. Incubation times and sdAb concentrations were selected based on previous optimization assays (data not shown). After incubation, cells were washed once with PBS and three times with TB. Each V L The translocation efficiency of the sdAbs was assessed by running the 15 ml collected volumes from the apical and basal sections on a 15% SDS-PAGE acrylamide gel followed by WB analysis using 1:3000 HRP-conjugated anti-His antibody (Roche). As a positive control, 100 ng of each purified V L sdAb was used. FC5 VHH was used as a positive control. The same procedure was performed to evaluate the BEB cross-crossing properties of each purified rabbit serum, but incubation was performed for 15 and 60 min and detection was performed with goat-α anti-rabbit IgG-Fc specific HRP at 1:10,000. Chemiluminescence was detected using a Chemidoc XRS+ System (Bio-rad). EXAMPLES

[0144] Biodistribution studies V selected for in vivo biodistribution study LsdAb by Cantante et al. (Cantante, C.; Lourenco, S.; Morais, M.; Leandro, J.; Gano, L.; Silva, N.; Leandro, P.; 858 Serrano, M.; Henriques, AO; Andre, A.; Cunha-Santos, C.; Fontes, C.; Correia, JDG; 859 Aires-da-Silva, F.; Goncalves, J. Albumin-Binding Domain from Streptococcus 860 Zooepidemicus Protein Zag as a Novel Strategy to Improve the Half-Life of Therapeutic 861 Proteins. J. Biotechnol. 2017, 253, 23-33. https: / / doi.org / 10.1016 / j.jbiotec.2017.05.017. 99 Mo / 99m The Na[ 99m TcO 4 A radioactive precursor [ 99m Tc(CO) 3 (H 2 O) 3 ] + was radiolabeled with

[0145] The radiochemical purity of the precursor was monitored by reversed-phase high-performance liquid chromatography (RP-HPLC) and instant thin-layer chromatography silica gel (ITLC-SG, Agilent Technologies, Santa Clara, CA, USA). 99m Tc(CO) 3 (H 2 O) 3 ] +The solution was added to a nitrogen-purged, sealed glass vial containing a solution of sdAb containing a His tag to give a final concentration of 1 mg / ml. The mixture was incubated at 37°C for 45-60 minutes. 99m Tc(CO) 3 The radiochemical purity of the -sdAb was assessed by ITLC-SG analysis using a 5% HCl (6 M) solution in MeOH as the eluent. 99m Tc(CO) 3 (H 2 O) 3 ] + and [TcO 4 ] - moves in front of the solvent (Rf=1), whereas radioactive sdAb, 99m Tc(CO) 3 -sdAb remains intact (Rf=0). Radioactivity distribution on the ITLC-SG strips was monitored using a miniGita Star scanning device (Raytest, Straubenhardt, DE) coupled with a Gamma BGO-V-Detector (Elysia Raytest, Straubenhardt, Germany). 99m Purification of Tc-labeled sdAb was performed using 10 K Amicon (Merck Millipore) centrifugal filters for protein purification and concentration as described by the supplier. The filtrate was discarded and 99m Tc(CO) 3 The concentrates containing -sdAbs were diluted in PBS and used for biodistribution studies in CD1 mice. Radiochemical purity (>95%) was determined by ITLC-SG. Biodistribution studies of radiolabeled sdAbs were performed as previously described [31, 45]. The corresponding sdAbs diluted in 100 ml PBS, pH 7.2 were injected into the tail vein of the animals. 99m Tc(CO) 3-sdAb (0.2-7.9MBq) was injected intravenously. Mice were sacrificed by cervical dislocation at 2 and 60 min after injection. The administered dose and radioactivity in sacrificed animals were measured using a dose calibrator (Carpintec CRC-15W). Differences between the radioactivity in injected and euthanized animals were assumed to be due to excretion. The desired brains and tissues were dissected, rinsed in PBS to remove excess blood, weighed, and their radioactivity was measured using a γ-counter (Berthold, Bad Wildbad, Germany). Uptake was calculated and expressed as percentage of injected radioactive dose per gram of organ or tissue (%ID / g). EXAMPLES

[0146] Measurement of brain antibody concentrations To verify the in vivo translocation efficiency of each rabbit serum, 100 μg of purified antibody was injected intravenously into the tail vein of CD1 female mice. Mice were sacrificed at 2 and 60 min after injection. Incubation times and antibody concentrations were selected based on previous optimization assays (data not shown). After blood collection, mouse brains, kidneys, and livers were isolated and homogenized as described above. Antibodies were recovered from each organ by immunoprecipitation (IP) with Dynabeads Protein A pull-down beads (rabbit serum) according to the manufacturer's protocol. 15 ml of IP eluate was separated in a 15% SDS PAGE gel and WB was performed with a 1:10,000 diluted conjugated anti-rabbit HRP antibody diluted 1:3000. Chemiluminescence was detected using a Chemidoc XRS+ System (Bio-rad). EXAMPLES

[0147] Development of PAN RG3 and FC5 functionalized liposomes Encapsulation of PAN into liposomes was performed by active loading using an ammonium sulfate gradient as previously described by Chen et al. (Chen, R.; Zhang, M.; Zhou, Y.; Guo, W.; Yi, M.; Zhang, Z.; Ding, Y.; Wang, Y. The 912 Application of Histone Deacetylases Inhibitors in Glioblastoma. J Exp Clin Cancer Res 913 2020, 39. https: / / doi.org / 10.1186 / s13046-020-01643-6.).

[0148] Briefly, the relevant lipids, dipalmitoylphosphatidylcholine (DPPC), poly(ethylene glycol) covalently linked to distearoylphosphatidylethanolamine (PEG-2000) (DSPE-PEG), and functionalized DSPE-PEG phospholipids containing biotin (DSPE-PEG-biotin) were dissolved in chloroform in a molar ratio of DPPC:Chol:DSPE-PEG:DSPE-PEG-biotin-1.85:1:0.14:0.01, purchased from Avanti Polar Lipids, and the organic solvent was removed by rotary evaporation. The homogeneous lipid film formed was hydrated with water, and the so-formed suspension was frozen (-70°C) and lyophilized overnight in a lyophilizer (Edwards, CO, USA). Rehydration of the lyophilized powder was performed with ammonium sulfate (135 mM, pH 5.4) at 45°C for 30 min. To produce a uniform liposome suspension, the unloaded liposomes were extruded (at 45°C) through a polycarbonate membrane of appropriate pore size using a Lipex 57 hermos-barrel extruder (Lipex: Biomembranes Inc., Vancouver, BC, Canada) under nitrogen pressure (10-500 lb / in) until liposomes with an average size of approximately 0.1 μm were achieved. 2) was filtered. An ammonium sulfate gradient was created by replacing excess liposome medium with PBS buffer (pH 7.4) using an Econo-pac 10 DG desalting column (Bio-Rad). PAN was incubated with unloaded liposomes pre-diluted in PBS (from a stock solution of 67 mg / ml) at a molar ratio of 1:16 μmol lipids for 60 min at 45°C. Unencapsulated PAN was separated by ultracentrifugation at 250,000×g for 2 h at 15°C in a Beckman LM-80 ultracentrifuge (Beckman Instruments, Inc., Fullerton, CA, USA). The pellet was suspended in PBS (pH 7.4). RG3 and FC5 antibodies were biotinylated at a molar ratio of 1:30 (molar antibody:molar biotin) using the kit EZ-Link™ Sulpho-NHS-LC-Biotinylation Kit (Thermo Fisher scientific). Biotin-antibody conjugates were mixed with streptavidin at a 3:1 molar ratio (mol antibody / mol streptavidin) for 20 min at room temperature. The mixture was then incubated with preformed biotin-liposomes at a 1:1 molar ratio (mol biotin in liposomes / mol biotinylated antibody) for 2 h at room temperature and then overnight at 4° C. Unbound sdAb was removed by centrifugation using a 100 K Amicon® Ultra-4 membrane filter (Merck). Biodistribution studies of selected RG3-conjugated PAN liposomes were performed using: 111 To this end, the chelator diethylenetriamine pentaacetic acid (DTPA) at a concentration of 6 μM was incorporated during the liposome preparation after achieving the lipid film and before lyophilization

[47] . The RG3-functionalized liposomes loaded with DTPA were used as precursors for the lipophilic complexes. 111 Using In-Oxine 111 Labeled with In. 111 The In-oxine complex passively crosses the lipid membrane and transfers the metal ion to DTPA in the inner aqueous compartment of the liposome, forming a hydrophilic complex.111 The In-DTPA remained trapped. Radiolabeling and subsequent biodistribution studies were carried out as described above. EXAMPLES

[0149] Translocation efficiency assay of Lip-RG3 and Lip-FC5 The translocation efficiency of each liposome formulation was verified on an in vitro BEB model using bEnd.3 cells as described above. Briefly, empty rhodamine-labeled RG3 and FC5-functionalized liposomes were pre-diluted to a final concentration of 1.15 ng / ml (taking into account the antibody ratio) in phenol red-free DMEM and added to the apical side of the in vitro BEB model. The apical and basal volumes were collected after 90 min, 6 and 24 h, and the fluorescence in these samples was measured separately in a microplate reader (Fluostar Optima Bmg Labtech) and the translocation was calculated using the formula: Translocation (%) = Fi / Ft x 100, where Fi is the fluorescence intensity recovered at the base and Ft is the fluorescence intensity of the total sdAb added to the apical side of the transwell. To determine the antitumor effects of PAN-encapsulated Lip-RG3 and Lip-FC5 on LN229 cells, cell viability assays were performed using the cell proliferation reagent WST-1 (Roche). 5 × 10 cells were cultured in 200 μL of DMEM culture medium supplemented with 10% FBS and 1% penicillin-streptomycin in a 96-well plate. 3Cells were seeded at a density of 1000 cells / well. Cells were subjected to increasing concentrations of PAN-encapsulated liposomes and respective controls (free PAN, Lip-PAN, Lip-RG3, Lip-FC5 and empty liposomes). After 24 hours of treatment, WST-1 reagent was added to determine cell viability according to the manufacturer's instructions. After 24 hours of incubation with the reagent, OD450nm was measured in a plate reader. Each data point was determined using triplicate wells and two independent experiments. Best fit IC50 values ​​were calculated using GraphPad Prism software (version 9.00, San Diego, CA, USA) using the logarithm (inhibitor) vs. response (variable slope) function. EXAMPLES

[0150] In vitro model of BEB-glioblastoma To test the BEB translocation efficiency of Lip-PAN-RG3 and Lip-PAN-FC5 liposomes and the subsequent drug delivery and cytotoxicity activity of PAN in glioblastoma cell lines, a dual in vitro non-contact co-culture model was established with bEnd.3 and LN229 cells. Briefly, bEnd.3 cells were cultured in tissue culture inserts as described above until confluence. 24 hours before the assay day, LN229 cells were cultured at 2 × 10 4 The bEnd.3 cells were cultured in 24-well plates at a density of 1000 x 1000 and the bEnd.3 cell cultures in the transwells were transferred to the 24-well plates. RG3 and FC5 functionalized liposomes loaded with free and encapsulated PAN were added to the apical side of the transwells and incubated for 24 hours. After incubation, the entire volume was withdrawn from the apical side, the transwells were removed, and the LN229 cells were incubated for an additional 24 hours. After 24 hours of treatment, WST-1 was added to the plates and after 24 hours of incubation, the OD 450nmwas measured. Each data point was determined using triplicate wells, and two independent experiments were performed on different days. To confirm that the cytotoxic effect of BBB-targeted PAN liposomes on GBM cell lines was associated with histone acetylation induction, protein extract samples were quantified using the Bradford method (Coomassie Plus™ Kit, Thermo Fisher Scientific) according to the manufacturer's instructions and analyzed by WB using anti-acetyl histone H3 (Lys9, Lys14) antibody (polyclonal, rabbit, 1:2500 dilution, Thermo Fisher Scientific), anti-histone H3 (polyclonal, rabbit, 1:1000 dilution, Thermo Fisher Scientific) as the primary antibody, and anti-rabbit IgG-Fc specific HRP (polyclonal, goat, 1:10,000 dilution, Jackson ImmunoResearch) as the secondary antibody. Protein detection was performed by chemiluminescence using Luminata Forte Western HRP (Merck) and acquired using a ChemiDoc XRS+ imaging system (Bio-Rad). Simultaneously, the integrity of the BEB model was measured as previously described. EXAMPLES

[0151] statistical analysis All data were expressed as mean ± standard error (SEM). Analyses were performed using Prism 9 (Graphpad Software). For in vitro assays, statistical significance of results was determined by one-way ANOVA followed by Tukey's multiple comparison test for comparing individual groups. Statistical analysis of biodistribution data (ANOVA for evaluation of data derived from labeled sdAb and t-test for data derived from liposomes) was also performed using GraphPad Prism (version 9.00) and the level of significance was set at a p-value of less than 0.05.

[0152] [Sequence table] Clone A12 SEQ ID NO:1 ELVMTQTPSSVSAAVGGTATIDCQSTKSVYNNNALSWYQQKPGQPPKLLIYRASTLTSGVPSRFSGSGSGTQFTLTINDVQCDDAATYYCQGGISGSSDAPFGGGGTEVVVK Clone C8 SEQ ID NO:2 ELVMTQTPSSVSAAVGGTVTINCRASEDIESYLAWYQQKPGQPPKRLIYGASNLASGVSSRFKGSGSGTDFTLTISDLECADAATYYCQCIVGGSTYGNVFGGGTELEIL Clone C5 SEQ ID NO:3 ELDLTQTPSPVSASVGGTATINCQASQSVYNNYWWAWYQQRPGHPPKLLIYETSKLASGVPSRFKGSGSGTQFTLTIIDVQCDDAATYVCAGGYGTVIDNAFGGGTELEIL Clone E1 SEQ ID NO:4 ELVMTQTPSPVSAAVGGTVTIACQSSQSVYSGTLLSWYQQKPGQSPKLLIHEASTLASGVPSRFKGSGSGTQFTLTISDLECADAATYYCLGGFDDDADNAFGGGTELEIL Clone E2 SEQ ID NO:5 ELDLTQTPPSLSASVGGTVTINCQASESISNYLAWYQQKPGQPPKLLIYGASNLESGVPSRFRGSGSGTEFTLTISDMKAEDAATFYCQSGYYTAGDLTFGAGTNVEIK Clone B12 SEQ ID NO:6 ELVLTQTPASVEAAVGGTVTIKCQASQSIGSYLAWYQQKPGQPPKLLIYGASTLASGVPSRFSGSRSDTQFSLTISDLECADAATYYCQGYYYSDSSSYVNTFGGGTEVVVK Clone NBC_482 SEQ ID NO:7 ELVMTQTPSSTSAAVGGTVTINCQSSPSVYSNNLAWYQQKPGHSPKLLIYKTSTLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCQGGYSDFIYGFGGGTELEIL Clone NBC_172 SEQ ID NO:8 ELVLTQTPSPVSAAVGGTVTINCQASQSVYNNNQLSWFQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTEYTLTISDVQCDDAATYYCQAYFNGYIWAFGGGTELEIL Clone NBC_94 SEQ ID NO:9 ELVLTQTPASVSEPVGGTVTIKCQASQSISTALAWYQQKPGQPPRLLIYDVSKLTSGVPSRFSGSGSGTEFTLTISDLECADAATYYCQCSADSSITNYGNAFGGGTELEIL Clone NBC_215 SEQ ID NO:10 ELVMTQTPSSVSAAVGGTVTIKCQASQNMASSLAWYQQKPGQPPKLLIYDASTLASGVPSRFKGGGSGTQFTLTITDLQCADAATYYCQSDYWTNAFGAFGGGTELEIL Clone NBC_286 SEQ ID NO:11 ELVLTQTPSPVSAAVGGTVTINCQASQSIGSDLSWYQQKPGQRPKLLIYDASTLASGVPSRFKGSGSGTEFTLTVSGVECADAATYYCQQGYSGTNVDNVFGGGTELEIL Clone NBC_431 SEQ ID NO:12 ELVLTQTPSSVSAAVGGTVTINCQASQSVWKNNELCWYQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTQFTLTINDVQCDGAATYYCLGTFNCSSADCNTFGGGTEVVVK Clone NBC_152 SEQ ID NO:13 ELVMTQTASSVSAAVGGTVTISCQASQSVYNDNWLSWYQQKLGQPPKQLIYRASNLETGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCAGGYSDEVFAFGGGTEVVVK Clone NC_1288 SEQ ID NO:14 ELVMTQTPSSVSAAVGGTATIDCQSTKSVYNNNALS*YQQKPGQPPKLLIYRASTLTSGVPSRFSGSGSGTQFTLTINDVQCDDAATYYCQGGISGSSDAPFGGGGTEVVVK Clone NC_268 SEQ ID NO:15 ELVLTQTPASVEVAVGGTVTINCQASQSIMNELSWYQQKPGQPPKLLIYRASTLTSGVPSRFSGSGSGTQFTLTINDVQCDDAATYYCQGGISGSSDAPFGGGGTEVVVK Clone NC_168 SEQ ID NO:16 ELVLTQTPASVEVAVGGTVTINCQASQSIMNELSWYQQKPGQPPKLLIYLASTLASGVPSRFKGSGSGTEFTLTITGVQCDDAATYYCQGGISGSSDAPFGGGGTEVVVK Clone NR_363 SEQ ID NO:17 ELVMTQTPSSSVSAAVGGTATIDCQSTKSVYNNNALSWYQQKPGQPPKLLIYRASTLTSGVPSRFSGSGSGTQFTLTINDVQCDDAATYCCQGGISGSSDAPFGGGGTEVVVK Clone NBC_67 SEQ ID NO:18 ELDLTQTPSSVSAAVGGTVTINCQSSQSVVSNKYLAWYQQKPGQPPMLLIYDVSTLASGVPSRFRGSGSGTQFTLTISDLECDDAATYYCQGSYAGSGYCSTFGGGTELEIL Clone NBC_70 SEQ ID NO:19 ELDMTQTPASVSEPVGGTVTIKCQASQSISSYLSWYQQKPGQPPKLLIYRASTLESGVPSRFKGSGSGTEYTLTISDLECADAATYYCQIYSSNYGYAFGGGTELEIL Clone NBC_72 SEQ ID NO:20 ELVLTQTPASVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYRASTLASGVPSRFKGSGSGTEFTLTISDLECADAATYYCQSYYGSSTTTYGNPFGGGTELEIL Clone NBC_73 SEQ ID NO:21 ELDLTQTPSSVSAAVGGTVTINCQASESISNYLAWYQQKPGQPPKLLIYGASNLESGVPSRFRGSGSGTEFTLTISDMKAEDAATFYCQSGYYTAGDLTFGAGTNVEIK Clone NBC_80 SEQ ID NO:22 ELDLTQTPPSLSASVGGTVTINCLASENVYSAVAWYQQKPEKPPTLLISGASNLESGVPPRFSGSGSGTDYTLTIGGVQAEDAATYFCQGYSSYPPTFGAGTNVEIK Clone NBC_81 SEQ ID NO:23 ELVLTQTPSSVSAAVGGTVSISCQASQSVYKNNYLSWFQQKPGQPPKLLIYDASTLASGVPSRFSGSGSGTEFTLTISDLECADAATYYCLGDDSSSSENVFGGGTELEIL Clone NBC_84_1 SEQ ID NO:24 ELVLTQTPSPVSAAVGGTVTISCQSSQSVYNNNYLSWFQQKPGQPPKLLIYETSKLESGVPSRFSGSGSGTQFTLTISDVQCDDAATYYCAGVYTTASDDSFGGGTEVVVK Clone NBC_84_2 SEQ ID NO:25 ELVMTQTPSSVEAPVGGTITIKCQASEDIYRLLAWYQQKPGQPPKLLIYDASTLASGVSSRFKGSGSGTEFTLTISDLECADGATYYCQSYYANSIGNAFGGGTEVVVK Clone NBC_85_1 SEQ ID NO:26 ELDLTQTPASVEAAVGGTVTINCRASEDIESLLAWYQQKPGQRPKLLIYDTSTLESGVPSRFKGSGSGTEYTLTISDLECDDAATYYCQSASYSSKSNPFGGGTELEIL Clone NBC_85_2 SEQ ID NO:27 ELVLTQTPSPVSAAVGGTVSISCQSSKNVYNNNYLAWYQQKPGQPPKLLIYTASSLASGVPSRFKGSGSGTQFSLTISDLECDDAATYYCAGGTGSTGDTAFGGGTEVVVK Clone NBC_86_1 SEQ ID NO:28 ELVMTQTPASVSEPVRGTVTIKCQASQNIRSWLSWYQQKPGQPPKLLIYQASKLASGVPSRFKGSGSETDFTLTISDLECADAATYYCQSNYDSSTTISYGADTFGGGTEVVVK Clone NBC_86_2 SEQ ID NO:29 ELVMTQTPASVSAAVGGTVTIKCQASQSVYGNNLLSWYQQKPGQPPKLLIYKASKLASGVPSRFSGSGSGTEFTLTISGVQCDDAATYYCLGVDYYQHNDGAAFGGGTEVVVK Clone NBC_87 SEQ ID NO:30 ELVLTQTPSPVSAAVGGTASISCQSSVSVYDNDRLSWYQQKPGQRPKLLIYAVSALASGVPSRFKGSGYGTQFTLTISDVQCDDAATYYCAGGYNDGFDGTFGGGTELEIL Clone NBC_88 SEQ ID NO:31 ELVLTQTPASMEVPVGGTVTITCQASESISSYLNWYQQKPGQPPKLLIFQASKLASGVPSRFKGSGSGTEFTLTISDVQCDDAATYYCQQGLINSNIDNTFGGGTEVVVK Clone NBC_89 SEQ ID NO:32 ELVLTQTPASVEVAVGGTVTINCQASQSIMNELSWYQQKPGQPPKLLIYLASTLASGVPSRFKGSGSGTEFTLTITGVQCDDAATYYCQQGYSHSNGAIDNVFGGGTELEIL Clone NBC_94_1 SEQ ID NO:33 ELDLTQTPASVEVAVGGTVTINCQASEDIGNLLAWYQQKPGQPPKLLIYKASTVASGVSSRFKGSGSGTQFTLTISGVECDDAATYYCQNNYGSSTIGHVYDFGGGTEVVVK Clone NBC_94_2 SEQ ID NO:34 ELVMTQTPASVEAAVGGTVTIKCQASQSIGTALAWYQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTQFTLTISDLECADAATYYCQGYDIGSGYGSNPFGGGTEVVVK Clone NBC_100_1 SEQ ID NO:35 ELDLTQTPASVEVPVGGTVTIKCQASQSISSELSWYQQQPGQRPKLLIYTAANLASGVPSRFKGSRSGTEFTLTISDLECADAATYYCQQGYSGDNVDNSFGGGTELEIL Clone NBC_100_2 SEQ ID NO:36 ELDLTQTPASVSEPVGGTVTIKCQASESVQNYLAWYQQKPGQPPKLLIYRASNLESGVSSRFKGSRSGTEFTLTISDLECADAATYYCQCTAGGNGYVGFGGGTELEIL Clone NBC_102 SEQ ID NO:37 ELVMTQTPASVSAAVGGTVTINCQASQSVYSNNYLSWYQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGYKSWNNDDFGFGGGTEVVVK Clone NBC_103 SEQ ID NO:38 ELVLTQTPASVSEPVGGTVTIKCQASQSINSWLSWYQQKPGQPPKLLIYQASKLASGVPSRFKGSGSGTEFTLTISDLECADAATYYCQNYYGISSYGRAFGGGTEVVVK Clone NBC_105 SEQ ID NO:39 ELVMTQTPSPVSAAVGGTVTISCQSSQSVDNNNRLAWYQQKVGQPPKLLIYKASTLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGDYKGNGDNDFGGGTELEIL Clone NBC_106 SEQ ID NO:40 ELVMTQTPASVSEPVGGTVTIKCQASQSIINLLAWYQQKPGQRPRLLMYSAFTLASGVPSRFKGSGSETEYTLTISDLECADAATYYCQSYYGGTYIAFGGGTELEIL Clone NBC_112_1 SEQ ID NO:41 ELVLTQTPSSSKSVPVGDTVTINCQASETVYDNNRLVWFQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTQFTLTISDVVCDDAATYYCAGYKSRGVDGSDFGGGTELEIL Clone NBC_132 SEQ ID NO:42 ELDLTQTPPSVSAAVGGTLTISCQSSESVYSNNRLSWYQQKPGQPPKLLIYEASTLSSGVPSRFKGSGSGTQFTLTISDVVCDDAATYYCQGNYYSSDWYNSFGGGTELEIL Clone NBC_141 SEQ ID NO:43 ELDLTQTPSSVSAAVGGTVTIKCQASQSVSSYLTWYQQKPGQPPKLLIYGASNLESGVPSRFRGSGSGTQFTLTISGMKAEDVATYYCHQHNSYPLTFGAGTNVEIK Clone RG3 SEQ ID NO:44 ELVMTQTPSPVSAAMGGTVSISCQSSKSVANNNELSWYQQKPGQPPKLLIYEASKLASGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCLGDYYSSSDNAFGGGTEVVVK Clone RG7 SEQ ID NO:45 ELVLTQTPSSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTEFLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK Clone RG15 SEQ ID NO:46 ELVXMXTQTPASVSAAVGGTVSISCQASQSVDWTTNLAWYQQKPGQRPKLLIYKASTLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCAGAATNTWAFGGGTELEIL Clone RG22 SEQ ID NO:47 ELVLTQTPASVEAAVGGTVTINCQASQSVYNNKNLAWYQQKPGQPPKLLIYRASTLASGVPSRFSGSGSGTQFTLTISGAQCDDAATYYCQGEFSCSSGDCIAFGGGTEVVVK Clone RG23 SEQ ID NO:48 ELVMTQTPASVSEPVGGTVTIKCQASQSIGNALAWYQQKPGQPPKLLIYGASNLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQSYYYSSSSSYGSYAFGGGTEVVVK Clone G27 SEQ ID NO:49 ELDMTQTPASVSAAVGGTVTIKCQASQTIGNYLAWYQQKPGQPPKLLISQASTLASGVPSRFRGSGSGTQFTLTISGVQCADAATYYCQCTDYGDTFGNAFGGGTEVVVK Clone G26 SEQ ID NO:50 ELVMTQTPASVSEAVGGTVTIKCQASQSIGGWLAWYQQKPGQRPKLLIYGASNLASGVPSRFSGSGSGTEFTLTISGVQCDDAATYYCQHGYTDTNIDNSFGGGTEVVVK Clone G25 SEQ ID NO:51 ELDMTQTPASVSEPVGGTVTIKCQASEGIYNNLVWYQQRPGQPPKLLIYRASTLASGVPSRFSGSGSGTHFTLTISDLECADAATYYCQSYYIRGSDFVNAFGGGGTEVVVK Clone G24 SEQ ID NO:52 ELVMTQTPSSASEPVGGTVTIKCQASESISSRLAWYQQKPGQRPKLLIYGASNLESGVPSRFKGSGSGTEYSLTISDLECDDAATYYCQSTYVSSTADTFAFGGGTEVVVK Clone G21 SEQ ID NO:53 ELDLTQTPASVEAAVGGTITINCQASESIDSSCASWYQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTQFSLTISGVRCDDAATYYCSGYKSYSNDDNGFGGGTEVVV Clone G20 SEQ ID NO:54 ELDMTQTPSSVSAAVGGTVTINCQASQSIGSWLAWYQQKPGQPPKQLIYDASTLASGVPSRFKGSGSGTEFTLTISGVQCDDAATYYCAGTYEISGWCGAFGGGTERRS Clone G19 SEQ ID NO:55 ELDMTQTPASVEAAVGGTVTIKCQASQNIYSNLAWYQQKPGQPPKLLIYEASRLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCLGAYNDDSDNTFGGTELEIL Clone G18 SEQ ID NO:56 ELDMTQTPSSTSEXVGGTVTIKCQASQNIGSDLSWYQQKPGQPPKLLIYQASKLASGVPSRFSGSRSRTEYTLAISGVQCDDAVTYYCLGVYGYSXEDGAAFGGGTELEIL Clone G17 SEQ ID NO:57 ELDMTQTPSSTSEPVGGTVTIKCQASQNIGSDLSWYQQKPGQPPKLLIYQASKLASGVPSRFSGSRSRTEYTLAISGVQCDDAVTYYCLGVYGYSSEDGAAFGGGTELEIL Clone G16 SEQ ID NO:58 ELDMTQTPASVSEPVGGTVTISCQSSQSVFDSNELSWYQQKPGQRPKPLIYDASKLASGVPSRFKGSGSGTRFTLTISDVQCDDAATYYCAGAFTTSSDNLFGGGTELEIL Clone G14 SEQ ID NO:59 ELVMTQTPASVEAAVGGTVTINCQASQSVYNNNRLAWYQQKPGQPPKLLIYYASDLASGVSSRFKGSGSGTQFTLTISDLECADAATYYCQTSYWRSSADTSAVFGGGTELEIL Clone G13 SEQ ID NO:60 ELVMTQTPPSLSASVGETVRIRCLASEDIYSGISWYQQKPGKPPTLLIYGASNLESGVPPRFSGSGSGTDYTLTIGGVQAEDAATYYCLGGYSYSSTGSLTFGAGTNVEIK Clone G12 SEQ ID NO:61 ELVLTQTPPSLXASVGETVRIRCLASEDIYSGISWYQQKPGKPPTLLISGAANLASGVPSRFSGSGSGTDYTLTIGGVQAEDAATYYCLGGWSYSDSGTTFGAGTNVEIK Clone G11 SEQ ID NO:62 ELVMTQTPSSVSAAVGGTVTINCQASKNINNYLAWYQQKPGQPPKLLIYGASNLESGVPSRFRGSGYGTEFTLTISDMKAEDAATYVCQSAYYSGGDLTFGAGTNVEIK Clone G9 / 10 SEQ ID NO:63 ELVMTQTPSSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTEFLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK Clone G8 SEQ ID NO:64 ELVLTQTPSSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTEFTLTIXGMNAEDAATYYCQSGYYSALTFGAGTNVEIK Clone G6 SEQ ID NO:65 ELVMTQTPSPVSAAVGGTVTIKCQSSQSVYNNNLLSWYQQKPGQPPKLLIYDASNLASGVPDRFSGSGSGTQFTLTISGVQCDDAATYYCLGGYDDDAEFGVGTNVEIK Clone G5 SEQ ID NO:66 ELVXTQTASPVSAAVGGTVTINCQSSQSVVDNNRLAWYQQKPGQPPKLLIYGASTLASGVPSRFKGSGSGTDFLTTISDVQCDDAATYYCLGTYVDDSHDAFGGGTEVVVK Clone G4 SEQ ID NO:67 ELVMTQTPSPVSAAVGGTVTISCQASQSVYSNYLSWFQQKPGQPPKLLIYGASTLASGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCAGGYATSSDNRAFGGGTEVVVK Clone G2 SEQ ID NO:68 ELVMTQTPSPVSAAVGGTVSINCQSSPSVYGSYLSWYQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTQFTLTISDVQCDDGATYYCAGGYSSGSDTGSAFGGGTEVVVK Clone G1 SEQ ID NO:69 ELVLTQTPSPVSAAVGGTVSISCQSSKSVYNNNWLSWFQQKPGQPPKQLIYYASTLASGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCAGGYSSSSDNLFGGGTELEIL Clone 60min_20 SEQ ID NO:70 ELDLTQTPSSTSEPVGGTVTIKCQASQSIGTKLSWYQQKPGQPPKLLIYQASKLASGVPSRFSGSGSGTDFTLTISDVQCDDAATYYWLGSYDCRSADCGAFGGGTEVVVK Clone 6_19 SEQ ID NO:71 ELVLTQTPSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTEFLTISGMKAEDAATYYCQSGYYSALTFGAGTNVDIK Clone 6_18 SEQ ID NO:72 ELDMTQTPSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTEFLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK Clone 6_17 SEQ ID NO:73 ELDMTQTPSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQLPKLLIYEASNLESGVPSRFRGSGSGTEFLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK Clone 6_16 SEQ ID NO:74 ELDLTQTPSSTSEPVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTEFLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK Clone 6_15 SEQ ID NO:75 ELVLTQTPSSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTEFLTISGMKAEDAATYYWQSGYYSALTFGAGTNVEIK Clone 6_14 SEQ ID NO:76 ELVMTQTPASVSEPVGGTVTIKCQASQSIGNALAWYQQKPGQPPKLLIYGASNLESGVPSRFRGSGSGTEFLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK Clone 6_13 SEQ ID NO:77 ELVLTQTASPVSAAVGGTVTINCQSSQSVVDNNRLAWYQQKPGQPPKLLIYGASTLASGVPSRFKGSGSGTDFTLTISDVQCDDAATYYCLGTYVDDSHDAFGGGTEVVVK Clone 6_12 SEQ ID NO:78 ELVMTQTPASVSEPVGGTVTIKCQASQSIGNALAWYQQKPGQPPKLLIYEASNLESGVPSRFRGGSGTEFTLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK Clone 6_11 SEQ ID NO:79 ELVLTQTPSSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTEFLTISGMKGEDAATYYCQSGYYSALTFGAGTNVEIK Clone 6_10 SEQ ID NO:80 ELVLTQTPSSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTEFLTISGMKAEDAATYYCQSGYYSALTFGAGTNVESK Clone 6_9 SEQ ID NO:81 ELDLTQTPSSTSEPVGGTVTIKCQASQSIGTKLSWYQQKPGQPPKLLIYEASNLESGVPSRFRGGSGTEFTLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK Clone 6_8 SEQ ID NO:82 ELVMTQTPSPVSAAMGGTVSISCQSSKSVANNNELSWYQQKPGQPPKLLIYEASKLASGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCLGDYYSSSDNAFGGGTEVVVK Clone 6_7 SEQ ID NO:83 ELVMTQTPSSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTEFLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK Clone 6_6 SEQ ID NO:84 ELDMTQTPSSTSEPVGGTVTIKCQASQNIGSDLSWYQQKPGQPPKLLIYQASKLASGVPSRFSGSRSRTEYTLAISGVQCDDAVTYYCLGVYGYSSEDGAAFGGGTELEIL Clone 6_5 SEQ ID NO:85 ELDLTQTPSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTEFLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK Clone 6_4 SEQ ID NO:86 ELVMTQTPASVSEPVGGTVTIKCQASQSIGNALAWYQQKPGQPPKLLIYGASNLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQSYYYSSSSSYGSYAFGGGTEVVVK Clone 6_3 SEQ ID NO:87 ELVMTQTPASVEAAVGGTVTIKCQASENIYSQLSWYQQKPGQRPKLLIYYASNLASGVSSRFTGSGSGTEYTLTISDLECADAATYYCQQGYTAGDVDNAFGGGTEVVVK Clone 6_2 SEQ ID NO:88 ELDLTQTPSSTSEPVGGTVTIKCQASQSIGTKLSWYQQKPGQPPKLLIYQASKLASGVPSRFSGSGSGTDFTLTISDVQCDDAATYYCLGSYDCRSADCGAFGGGTEVVVK Clone 6_1 SEQ ID NO:89 ELVLTQTPSSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTEFLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK Clone 5_20 SEQ ID NO:90 ELVLTQTASPVSAAVGGTVTINCQSSQSVVDNNRLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTEFLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK Clone 5_19 SEQ ID NO:91 ELVLTQTPSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTQFTLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK Clone 5_18 SEQ ID NO:92 ELVMTQTPSPVSAAMGGTVSISCQSSKSVANNNELSWYQQKPGQPPKLLIYEASNLESGVPSRFRGGSGTEFTLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK Clone 5_17 SEQ ID NO:93 ELVMTQTPSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTQFTLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK Clone 5_16 SEQ ID NO:94 ELVMTQTPASVSAAVGGTVTINCQASQSLYNNKNLAWYQQKPGQPPKRLMYATSNLASGVSSRFKGSGSGTQFTLTISDLECADAATYYCQSPFYGSGDVFAFGGGTELEIL Clone 5_15 SEQ ID NO:95 ELVMTQTPASVSEPVGGTVTIKCQASQSIGNALAWYQQKPGQPPKLLIYGASNLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQSYYYSSSSSYGSYAFGGGTEVVVK Clone 5_14 SEQ ID NO:96 ELVMTQTPASVSEPVGGTVTINCQASQSIGSNLAWYQQKAGQPPKLLIYYASTLESGVPSRFSGSGSGTQFTLTISDVQCDDAATYYCAGADTSSADSAFGGGTELEIL Clone 5_13 SEQ ID NO:97 ELVLTQPQSVSGSLGQTVSISCNRDSGNIEDYYVHWYQQHPGKAPTTVVYNDDQRPSGVPDRFSGSIDSTSNSASLTITDLLAEDEADYYCLSSDSSANVFGGGTQLTVTG Clone 5_12 SEQ ID NO:98 LDMTQTPSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTEFTLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK Clone 511 SEQ ID NO:99 ELVMTQTPASVEAAVGGTVTINCQASQSVYNNKNLAWYQQKPGQPPKLLIYRASTLASGVPSRFSGSGSGTQFTLTISGAQCDDAATYYCQGEFSCSSGDCIAFGGGTEVVVK Clone 5_10 SEQ ID NO:100 ELDMTQTPSSTSEPVGGTVTIKCQASQNIGSDLSWYQQKPGQPPKLLIYQASKLASGVPSRFSGSRSRTEYTLAISGVQCDDAVTYYCLGVYGYSSEDGAAFGGGTELEILG Clone 5_9 SEQ ID NO:101 ELVMTQTPASVSAAVGGTVSISCQASQSVDWTTNLAWYQQKPGQRPKLLIYKASTLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCAGAATNTWAFGGGTELEIL Clone 5_8 SEQ ID NO:102 ELDLTQTPASVEAAVGGTITINCQASESIDSSCASWYQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTQFSLTISGVRCDDAATYYCSGYKSYSNDDNGFGGGTEVVVK Clone 5_7 SEQ ID NO:103 ELVLTQTPSSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYDASNLESGVPSRFRGSGSGTEFLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIKG Clone 5_6 SEQ ID NO:104 ELDLTQTPSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTQFTLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK Clone 5_5 SEQ ID NO:105 ELVLTQTASPVSAAVGGTVTINCQSSQSVVDNNRLAWYQQKPGQPPKLLIYGASTLASGVPSRFKGSGSGTDFTLTISDVQCDDAATYYCLGTYVDDSHDAFGGGTEVVVK Clone 5_4 SEQ ID NO:106 ELVMTQTPSPVSAAMGGTVSISCQSSKSVANNNELSWYQQKPGQPPKLLIYEASKLASGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCLGDYYSSSDNAFGGGTEVVVK Clone 5_3 SEQ ID NO:107 ELVMTQTPSSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTEFLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK Clone 5_2 SEQ ID NO:108 ELDLTQTPSSVSAAVGGTVTINCQASQSISNLLAWYQQKPGQPPKLLIYEASNLESGVPSRFRGSGSGTEFLTISGMKAEDAATYYCQSGYYSALTFGAGTNVEIK

Claims

1. An antibody-drug conjugate (ADC) for drug delivery having antitumor properties, comprising a single-domain antibody (sdAb) conjugated to an antitumor payload agent, wherein the conjugation of the payload is in the free, exposed cysteine at position 80, 23, or 88 of the VL unit, the single-domain antibody (sdAb) is a VL unit modified to exhibit a single free cysteine, and the VL unit comprises a rabbit sdAb having at least one sequence selected from SEQ ID NOs: 1 to 43, said antibody-drug conjugate (ADC).

2. The antibody-drug conjugate (ADC) according to claim 1, wherein the VL chain comprises a rabbit sdAb having at least one sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6.

3. The antibody-drug conjugate (ADC) according to claim 1, wherein the antitumor payload agent is an SN38 molecule.

4. The antibody-drug conjugate (ADC) according to any one of claims 1 to 3, wherein the antitumor payload agent SN38 molecule further comprises maleimide conjugated by a diazaborine bioconjugation linker.

5. A pharmaceutical composition comprising the antibody-drug conjugate (ADC) according to any one of claims 1 to 3, further comprising an acceptable pharmaceutical agent.

6. The pharmaceutical composition according to claim 5, for use as a medicament in tumor therapy.

7. The tumor is a solid tumor, blood, or lymphatic tumor, preferably, the tumor is breast cancer, triple-negative breast cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, gastrointestinal cancer, acute myeloid leukemia, multiple myeloma, cervical cancer, lung cancer, prostate cancer, colorectal cancer, ovarian cancer, kidney cancer, or thyroid cancer, the pharmaceutical composition according to claim 6.

8. The pharmaceutical composition according to claim 6, wherein the tumor is present in a mammal, preferably, a human or canine tumor.

9. A method for obtaining an antibody-drug conjugate (ADC), comprising the following steps: a) preparing lymph node primary cells derived from a canine multicentric lymphoma biobank; b) A step of immunizing 1 x 10 lymph node primary cells ７ derived from the canine multicentric lymphoma biobank into rabbits, c) isolating RNA and cDNA from samples of spleen and bone marrow; d) constructing a single-domain antibody sdAB targeting cNHL and hNHL according to claim 1 or 2; e) V according to claim 1 or 2 Ｌ V having a free, exposed cysteine at position 80, 23 or 88 of the framework Ｌ synthesizing a rabbit sdAb in the format of the light chain variable region f) binding VL to cNHL and hNHL cells by incubation with anti-HA FITC antibody; g) conjugating VL with DAB-SN38 by adding a solution of DAB-SN38 to the solution of TCEP and VL; The method as described above.

10. A drug delivery system for targeting BBB endothelial cell receptors in the central nervous system, comprising a rabbit-derived single domain antibody (sdAb) conjugated to the surface of liposomes encapsulating a suitable drug that enables efficient blood-brain barrier (BBB) translocation, wherein the sdAb is defined by SEQ ID NO: 44 to SEQ ID NO:

108. The drug delivery system.

11. The drug delivery system according to claim 10, wherein the sdAb is defined by SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, which are RG3, RG7, RG15, RG22 and RG23 respectively.

12. The drug delivery system according to claim 10 or 11, wherein the liposomes conjugated with sdAb on the surface are SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO:

48.

13. The drug delivery system according to claim 10 or 11, wherein the suitable drug is a pan histone deacetylase inhibitor (PAN).

14. A pharmaceutical composition comprising the drug delivery system according to claim 10 or 11, further comprising an acceptable pharmaceutical agent.

15. A method for producing the drug delivery system according to claim 10 or 11, comprising the following steps: a) preparing a rabbit immunized with a cell line of mouse brain endothelial cells (bEnd.3); b) recovering a rabbit antibody capable of passing through the BBB barrier from the serum of the immunized rabbit described in a); c) preparing an sdAb library, wherein the sdAb is derived from an antibody light chain variable region (V Ｌ ), and the sdAb library is obtained by cloning the V Ｌ sdAb region into a pComb3X phagemid vector; d) selecting an sdAb presented on the surface of a phage having target specificity characteristics of blood-brain barrier (BBB) translocation; e) conjugating the sdAb to the surface of liposomes; f) encapsulating a suitable drug that enables efficient blood-brain barrier (BBB) translocation into the conjugated liposomes of e); The method as described above.

16. The method according to claim 15, wherein the sdAb of (d) is further processed by injecting the sdAb composition into CD1 mice, recovering phages from the mouse brain, and collecting brain-specific sdAbs.

17. The process is repeated once, preferably twice, more preferably three times, resulting in an improved ability to reach the BBB, 10 ５ The method according to claim 16, wherein a concentrated phage having a titer (phage / mL) of

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