CAR-T cell therapy is at the forefront of next-generation multiple myeloma (MM) management, with two B-cell maturation antigen (BCMA)-targeted products recently approved. However, these products are ...incapable of breaking the infamous pattern of patient relapse. Two contributing factors are the use of BCMA as a target molecule and the artificial scFv format that is responsible for antigen recognition. Tackling both points of improvement in the present study, we used previously characterized VHHs that specifically target the idiotype of murine 5T33 MM cells. This idiotype represents one of the most promising yet challenging MM target antigens, as it is highly cancer- but also patient-specific. These VHHs were incorporated into VHH-based CAR modules, the format of which has advantages compared to scFv-based CARs. This allowed a side-by-side comparison of the influence of the targeting domain on T cell activation. Surprisingly, VHHs previously selected as lead compounds for targeted MM radiotherapy are not the best (CAR-) T cell activators. Moreover, the majority of the evaluated VHHs are incapable of inducing any T cell activation. As such, we highlight the importance of specific VHH selection, depending on its intended use, and thereby raise an important shortcoming of current common CAR development approaches.
HER2-targeted therapies have drastically improved the outcome for breast cancer patients. However, when metastasis to the brain is involved, current strategies fail to hold up to the same promise. ...Camelid single-domain antibody-fragments (sdAbs) have been demonstrated to possess favorable properties for detecting and treating cancerous lesions in vivo using different radiolabeling methods. Here we evaluate the anti-HER2 sdAb 2Rs15d, coupled to diagnostic γ- and therapeutic α- and β
-emitting radionuclides for the detection and treatment of HER2
brain lesions in a preclinical setting. 2Rs15d was radiolabeled with
In,
Ac and
I using DTPA- and DOTA-based bifunctional chelators and Sn-precursor of SGMIB respectively and evaluated in orthotopic tumor-bearing athymic nude mice. Therapeutic efficacy as well as systemic toxicity were determined for
I- and
Ac-labeled sdAbs and compared to anti-HER2 monoclonal antibody (mAb) trastuzumab in two different HER2
tumor models. Radiolabeled 2Rs15d showed high and specific tumor uptake in both HER2
SK-OV-3-Luc-IP1 and HER2
MDA-MB-231Br brain lesions, whereas radiolabeled trastuzumab was unable to accumulate in intracranial SK-OV-3-Luc-IP1 tumors. Administration of
I-2Rs15d and
Ac-2Rs15d alone and in combination with trastuzumab showed a significant increase in median survival in 2 tumor models that remained largely unresponsive to trastuzumab treatment alone. Histopathological analysis revealed no significant early toxicity. Radiolabeled sdAbs prove to be promising vehicles for molecular imaging and targeted radionuclide therapy of metastatic lesions in the brain. These data demonstrate the potential of radiolabeled sdAbs as a valuable add-on treatment option for patients with difficult-to-treat HER2
metastatic cancer.
Multiple myeloma (MM) remains incurable, despite the advent of chimeric antigen receptor (CAR)-T cell therapy. This unfulfilled potential can be attributed to two untackled issues: the lack of ...suitable CAR targets and formats. In relation to the former, the target should be highly expressed and reluctant to shedding; two characteristics that are attributed to the CS1-antigen. Furthermore, conventional CARs rely on scFvs for antigen recognition, yet this withholds disadvantages, mainly caused by the intrinsic instability of this format. VHHs have been proposed as valid scFv alternatives. We therefore intended to develop VHH-based CAR-T cells, targeting CS1, and to identify VHHs that induce optimal CAR-T cell activation together with the VHH parameters required to achieve this.
CS1-specific VHHs were generated, identified and fully characterized,
and
. Next, they were incorporated into second-generation CARs that only differ in their antigen-binding moiety. Reporter T-cell lines were lentivirally transduced with the different VHH-CARs and CAR-T cell activation kinetics were evaluated side-by-side. Affinity, cell-binding capacity, epitope location,
behavior, binding distance, and orientation of the CAR-T:MM cell interaction pair were investigated as predictive parameters for CAR-T cell activation.
Our data show that the VHHs affinity for its target antigen is relatively predictive for its
tumor-tracing capacity, as tumor uptake generally decreased with decreasing affinity in an
model of MM. This does not hold true for their CAR-T cell activation potential, as some intermediate affinity-binding VHHs proved surprisingly potent, while some higher affinity VHHs failed to induce equal levels of T-cell activation. This could not be attributed to cell-binding capacity,
VHH behavior, epitope location, cell-to-cell distance or binding orientation. Hence, none of the investigated parameters proved to have significant predictive value for the extent of CAR-T cell activation.
We gained insight into the predictive parameters of VHHs in the CAR-context using a VHH library against CS1, a highly relevant MM antigen. As none of the studied VHH parameters had predictive value, defining VHHs for optimal CAR-T cell activation remains bound to serendipity. These findings highlight the importance of screening multiple candidates.
Multiple myeloma (MM) is a hematological malignancy characterized by the presence of clonal plasma cells in the bone marrow niche. Despite significant therapeutic advances, MM remains incurable for ...the majority of patients. Targeted radionuclide therapy (TRNT) has emerged as a promising treatment option to eradicate residual cancer cells. In this study, we developed and characterized single-domain antibodies (sdAbs) against the MM-antigen CS1 and evaluated its therapeutic potential in MM using TRNT. We first validated CS1 as potential target for TRNT. CS1 is expressed in normal and malignant plasma cells in different disease stages including progression and relapse. It is expressed in dormant as well as proliferating MM cells, while low expression could be observed in environmental cells. Biodistribution studies demonstrated the specific uptake of anti-CS1 sdAbs in tissues of 5TMM cell infiltration including bone, spleen and liver. TRNT using anti-CS1 sdAbs labeled with actinium-225 significantly prolonged survival of syngeneic, immunocompetent 5T33MM mice. In addition, we observed an increase in CD8
+
T-cells and more overall PD-L1 expression on immune and non-immune cells, implying an interferon gamma signature using actinium-225 labeled CS1-directed sdAbs. In this proof-of-principle study, we highlight, for the first time, the therapeutic potential and immunomodulating effects of anti-CS1 radionuclide therapy to target residual MM cells.
Single domain antibodies (sdAbs) have proven to be valuable probes for molecular imaging. In order to produce such probes, one strategy is the functionalization of the reactive amine side chain of ...lysines with a chelator, resulting in a mixture of compounds with a different degree of conjugation. In this study, we implemented anion exchange chromatography (AEX) to separate the different compounds or fractions that were further characterized and evaluated to study the impact of the conjugation degree on pharmacokinetic properties and functionality. Anti-HER2 and anti-MMR sdAbs were functionalized with NOTA or DTPA chelator. Anion exchange chromatography was performed using 0.02 mol/L Tris pH 7.5 as the first solvent and 0.25 M or 0.4 M NaCl (in case of NOTA chelator or DTPA chelator, respectively) as the second solvent applied as a gradient. The fractions were characterized via mass spectrometry (MS), surface plasmon resonance (SPR), and isoelectric focusing gel electrophoresis (IEF), while in vivo studies were performed after radiolabeling with either
Ga (NOTA) or
In (DTPA) to assess the impact of the conjugation degree on pharmacokinetics. AEX could successfully be applied to separate fractions of (chelator)
-anti-HER2 and (chelator)
-anti-MMR sdAb constructs. MS confirmed the identity of different peaks obtained in the separation process. SPR measurement suggests a small loss of affinity for (chelator)
-anti-sdAb, while IEF revealed a correlated decrease in isoelectric point (pI) with the number of conjugated chelators. Interestingly, both the reduction in affinity and in pI was stronger with the DTPA chelator than with NOTA for both sdAbs. In vivo data showed no significant differences in organ uptake for any construct, except for (DTPA)
-anti-MMR, which showed a significantly higher liver uptake for (DTPA)
-anti-MMR compared to (DTPA)
-anti-MMR and (DTPA)
-anti-MMR. For all constructs in general, high kidney uptake was observed, due to the typical renal clearance of sdAb-based tracers. The kidney uptake showed significant differences between fractions of a same construct and indicates that a higher conjugation degree improves kidney clearance. AEX allows the separation of sdAbs with a different degree of conjugation and provides the opportunity to further characterize individual fractions. The conjugation of a chelator to sdAbs can alter some properties of the tracers, such as pI; however, the impact on the general biodistribution profile and tumor targeting was minimal.
Immuno‐oncology has been at the forefront of cancer treatment in recent decades. In particular immune checkpoint and chimeric antigen receptor (CAR)‐T cell therapy have achieved spectacular results. ...Over the years, CAR‐T cell development has followed a steady evolutionary path, focusing on increasing T cell potency and sustainability, which has given rise to different CAR generations. However, there was less focus on the mode of interaction between the CAR‐T cell and the cancer cell; more specifically on the targeting moiety used in the CAR and its specific properties. Recently, the importance of optimizing this domain has been recognized and the possibilities have been exploited. Over the last 10 years—in addition to the classical scFv‐based CARs—single domain CARs, natural receptor‐ligand CARs, universal CARs and CARs targeting more than one antigen have emerged. In addition, the specific parameters of the targeting domain and their influence on T cell activation are being examined. In this review, we concisely present the history of CAR‐T cell therapy, and then expand on various developments in the CAR ectodomain. We discuss different formats, each with their own advantages and disadvantages, as well as the developments in affinity tuning, avidity effects, epitope location, and influence of the extracellular spacer.
Targeted therapy and immunotherapy have become mainstream in cancer treatment. However, only patient subsets benefit from these expensive therapies, and often responses are short-lived or coincide ...with side effects. A growing modality in precision oncology is the development of theranostics, as this enables patient selection, treatment and monitoring. In this approach, labeled compounds and an imaging technology are used to diagnose patients and select the best treatment option, whereas for therapy, related compounds are used to target cancer cells or the tumor stroma. In this context, nanobodies and nanobody-directed therapeutics have gained interest. This interest stems from their high antigen specificity, small size, ease of labeling and engineering, allowing specific imaging and design of therapies targeting antigens on tumor cells, immune cells as well as proteins in the tumor environment. This review provides a comprehensive overview on the state-of-the-art regarding the use of nanobodies as theranostics, and their importance in the emerging field of personalized medicine.
To this day, multiple myeloma remains an incurable cancer. For many patients, recurrence is unavoidably a result of lacking treatment options in the minimal residual disease stage. This is due to ...residual and treatment-resistant myeloma cells that can cause disease relapse. However, patient-specific membrane-expressed paraproteins could hold the key to target these residual cells responsible for disease recurrence. Here, we describe the therapeutic potential of radiolabeled, anti-idiotypic camelid single-domain antibody fragments (sdAbs) as tumor-restrictive vehicles against a membrane-bound paraprotein in the syngeneic mouse 5T33 myeloma model and analogously assess the feasibility of sdAb-based personalized medicine for patients with multiple myeloma. Llamas were immunized using extracts containing paraprotein from either murine or human sera, and selective sdAbs were retrieved using competitive phage display selections of immune libraries. An anti-5T33 idiotype sdAb was selected for targeted radionuclide therapy with the β
-particle emitter
Lu and the α-particle emitter
Ac. sdAb-based radionuclide therapy in syngeneic mice with a low 5T33 myeloma lesion load significantly delayed tumor progression. In five of seven patients with newly diagnosed myeloma, membrane expression of the paraprotein was confirmed. Starting from serum-isolated paraprotein, for two of three selected patients anti-idiotype sdAbs were successfully generated.
Enterotoxemia is characterized by a highly fatal hemorrhagic enteritis in cattle, caused by Clostridium perfringens. Production systems with intensive feeding, such as the veal industry, are ...predisposed. The primary objective of this study was to determine the effect of solid feed provision on fecal C. perfringens excretion in veal calves. Ten Holstein Friesian bull veal calves were randomly assigned to one of two test diets. Group I received solemnly milk replacer twice daily, while group 2 received milk replacer and a maximum of 300g solid feed/day, consisting of a mixture of 30 barley, 30 corn, 30 hulled wheat and 10 chopped straw. The number of C. perfringens per g feces or fecal clostridia! counts (FCC) were determined for all calves. Mean FCC were significantly lower in the calves fed milk replacer and solid feed, than in the calves fed solemnly milk replacer. Although the correlation between FCC and enterotoxemia risk remains to be determined, the provision of solid feed to veal calves reduced clostridial excretion, which might contribute to the prevention of this disease.
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