Nanobodies: a new potential for prostate cancer treatment

Abstract

Background

The current progressive increase in the cancer burden of prostate cancer requires the exploration of new diagnostic and therapeutic approaches. Nanobodies are single-domain antibodies with the advantages of small size, high stability, easy processing and modification, which are increasingly used in the treatment of many types of cancer.

Methods

This review analyzed the relevant literature in PubMed and other databases.

Result

In the retrieved literature, nanobodies are widely used in the treatment of prostate cancer. The preparation of nanobodies targeting PSA or PSMA is straightforward. For diagnostic purposes, nanobodies can be used in the preparation of biosensors for more sensitive identification of prostate cancer; for therapeutic purposes, nanobodies are used in the preparation of immunotoxic and ADC drugs. Preclinical in vivo and in vitro experiments have shown that this therapeutic approach is feasible. This article is a review of the above to provide new ideas for the treatment of prostate cancer.

Conclusion

Compared with traditional antibodies, nano-antibodies have the advantages of small size, high stability, and high penetration. These advantages make nano-antibodies worthy to be widely used. Current studies have shown that nanobodies have advantages and future in the diagnosis and treatment of prostate cancer.

Introduction

According to epidemiological data (Siegel et al. 2022), prostate cancer accounts for 27% of new cancers in men and is the second most common cancer among men. Although the androgen deprivation-based treatment paradigm has prolonged patient survival as much as possible, the lack of effective treatment at advanced stages still leads to poor prognosis. Therefore, in the face of its increasing cancer burden, we must actively explore new therapeutic approaches.

Currently, various polyclonal antibodies (pAb) and monoclonal antibodies (mAb) are increasingly becoming an indispensable part of basic medical research, clinical diagnosis, and treatment. Taking mAb as an example, various immune checkpoint inhibitors of mAb are approved for the treatment of non-small cell lung cancer and other malignant tumors, which have considerably changed the treatment landscape of malignant tumors (Bagchi et al. 2021). However, it remains unsatisfactory that conventional pAb and mAb have limitations in terms of their solubility, stability, and permeability (Bannas et al. 2017). In contrast, nanobodies (Nbs) have emerged to some extent to compensate for the deficiencies of traditional antibodies. They can be used as antitoxic agents, antitoxic agents, antiviral agents, antibacterial agents, antiparasitic agents, anti-inflammatory agents, immunomodulators, anticancer agents, antifungal agents, and biosensor tools, among others, bringing new directions in the development of biomedicine (Mir et al. 2020).

Brief description of nanobodies.

Antibodies are the central molecules of the biological immune system, capable of recognizing and removing pathogens that invade the organism. Immunoglobulin G (IgG) is a protein with a molecular weight of 150 kDa, which usually consists of two identical heavy chains covalently linked by disulfide bonds and two identical light chains (Fig. 1a). The presence of this complex structure of inter- and intra-molecular disulfide bonds makes it susceptible to intracellular redox environments and payloads; at the same time, the antigen-binding domain (CDR) of IgG contains a highly conserved loop length, which limits the range of antigen binding. These reasons have led to deficiencies in the production, manufacture, functionalization, and intracellular application of conventional antibodies, and therefore, a variety of recombinant antibody products, including nanobodies, that break through these limitations are being developed and widely used in the treatment of a variety of diseases (Schumacher et al. 2018).

Fig. 1.

a Monoclonal antibodies. VH variable heavy-chain domain, VL variable light-chain domain, CH constant heavy-chain domain, CL constant light-chain domain. b VHH: variable domain of heavy chain-only antibody

In the early 1990s, Hamers-Casterman et al. detected a specific class of heavy chain antibodies (hcAb) in the sera of Camelidae (Hamers-Casterman et al. 1993). These hcAbs do not have the light chain structure of conventional antibodies, and the functional antigen-binding unit is reduced to a single variable structural domain (Fig. 1b). This single variable domain structure has a molecular weight of approximately 13–14 kDa It is therefore commonly referred to as a nano- or single-domain antibody (Nb/VHH), the smallest antibody unit obtained to date that can bind antigens stably and fully. Compared to conventional antibodies, nanobodies have a longer CDR3 structure that allows them to bind antigens in a convex complementary position (De Genst et al. 2006); the hydrophobic amino acids responsible for the VH/VL interaction in the antibody backbone region (FR) are replaced by hydrophilic amino acids, which allows them to have better solubility (Maass et al. 2007). In addition, nanobodies are characterized by high stability (Allegra et al. 2018), low immunogenicity (De Munter et al. 2020), strong tissue penetration (Travis McMurphy 2014), and ease of modification.

1. Compared with mAb, Nb has a small molecular weight while retaining antigen binding and has the ability to penetrate dense tissues

2. Compared to VH, the CDR3 loop in Nb extends outward and binds with high affinity to the concave epitope of the protein active site.

3. The complementary positions of CDR1 and CDR2 in Nb contain more hydrophobic amino acids, and residues in the backbone region are also involved in antigen binding.

Progress in the application of nanoantibodies in the diagnosis and treatment of prostate cancer

Due to the above-mentioned advantages of nanobodies, it is used in diagnosing and treating various cancers, and several clinical trials are already underway (see Table 1). To further explore its research in prostate cancer, we reviewed the relevant literature in PubMed, Scopus, and Web of Science.

Table 1.

Registered clinical studies of nanobodies in the field of oncology (Chinese Clinical Trial Registry And https://clinicaltrials.gov/)

	Scientific title	Tumor	Phase
1	A Novel VHH-Based Radiotracer, 99mTc-Mirc324, for SPECT/CT Imaging of GPC3-Positive Hepatocellular Carcinoma Patients	Hepatic-cellular-carcinoma	Not applicable
2	Clinical study on the immunophenotypic classification of autocrine PD-1 nano antibody MesO-CAR T combined with apatinib in the treatment of advanced gastric cancer	Gastric cancer	Phase 0
3	An exploratory study of aPD1-MSLN-CAR T cells for the treatment of MSLN-positive advanced solid tumors	Advanced solid tumors	Phase 0
4	Multiple Ascending Dose Trial of MSB0010841(Anti-IL17A/F Nanobody) in Psoriasis Subjects	Solid tumor	Not applicable
5	PD-L1 Targeting Nanobody Probe for PET Imaging of Solid Tumor	Lung cancer melanoma	Not applicable
6	CD19/20 Bispecific Nanobody-derived CAR-T Cells in B Cell Lymphoma	B-Cell lymphoma stage I	Phase 1
7	αPD1-MSLN-CAR T Cells for the Treatment of MSLN-positive Advanced Solid Tumors	Non-small-cell-lung cancer mesothelioma	Phase 1
8	BCMA-CAR-T in Relapsed/Refractory Multiple Myeloma	Relapsed/refractory myeloma	Phase 1
9	αPD1-MSLN-CAR T Cells for the Treatment of MSLN-positive Advanced Solid Tumors	Colorectal cancer ovarian cancer	Phase 1

Data from Chinese Clinical Trial Registry And https://clinicaltrials.gov/

Application of nanobodies in prostate cancer diagnosis

The most basic function of antibodies is to specifically bind to and activate the antigen catabolic process (Goulet and Atkins 2020), This targeting provides the basis for diagnosing and treating prostate cancer with nanobodies. In the literature we included, most of the nanobodies they studied were designed against prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA).

Application of nanobodies targeted PSA

PSA consists of a chymotrypsin serine protease encoded by the KLK3 gene, related studies have shown (Moradi et al. 2019) that PSA is essential in maintaining cancer cell growth, mediating epithelial-mesenchymal transition (EMT), invasion and metastasis, inducing osteoblast differentiation, and promoting tumor angiogenesis. These characteristics make PSA, the most important biomarker for prostate cancer as it can be used for screening, risk of recurrence stratification, post-diagnostic surveillance and all major stages of treatment of prostate cancer patients (Duffy 2020).

Because of the importance of PSA in the diagnosis and detection of prostate cancer, we expect that the application of nanobodies to PSA will be able to recognize PSA and its related products more sensitively. The work of Dirk Saerens et al. laid the groundwork for this vision (Saerens et al. 2004) by using peripheral blood and lymph node lymphocytes from PSA-immunized dromedary camels by hybridoma technique to generate two VHH gene libraries and retrieved multiple VHHs capable of binding PSA from both libraries. The various VHHs showed a wide range of kinetic rate constants for free PSA, with the best nanoantibody having a Kd value of 0.16 nm for N7. More interestingly, the same VVH showed different Kd values for different PSA isomers, taking N7 as an example, it showed Kd values of 0.16, 0.18, and 0.18 nm for free PSA, PSA non-ACT-binding, and PSA-ACT complex, respectively. we know that different PSA isomer levels are indicative of the prognosis of prostate cancer Therefore, VHHs can sense the structural changes of different PSA isoforms, and this feature can be used for the more accurate clinical judgment of patients.

The work of Dirk Saerens et al. confirmed that nanobodies have a selective effect on PSA, and how to further apply this property to serve the clinic became a question we had to think about, and biosensors entered the vision of researchers. A biosensor is a small analytical device that combines a biometric system and a physicochemical sensor to detect a target molecule by converting the recognized signal into a detectable output signal (Roointan et al. 2019). Since the sensitivity of a biosensor depends on the bioreceptor on its surface, nanobodies are considered an ideal probe under their small size, high affinity, specificity, and stability (Bastos-Soares et al. 2020). Anke K. Trilling et al. used nanobodies to construct a biosensor for foot-and-mouth disease They found that compared to a randomly oriented biosensor, the uniformly oriented biosensor had detection limit was reduced by 800-fold compared to randomly oriented biosensors (Trilling et al. 2014).

S-layers are protein crystal arrays formed on the outermost envelope of bacteria with molecular weights of 40–200 kDa and thicknesses of 5–25 nm, which can play a role in maintaining bacterial cell morphology and are widely used in magnetic ultrafiltration membrane production, biosensor preparation and multifunctional nanoparticle drug delivery system construction due to their excellent stability and adhesion (Luo et al. 2019).Magdalena Pleschberger et al. (Pleschberger et al. 2004) prepared an S-layer fusion protein for the generation of nanopatterned sensing layers for the detection of PSA by surface plasmon resonance (SPR) technique using Bacillus spherics CCM 2177. The S-layer protein molecule consists of a secondary cell wall polymer (SCWP) as the N-terminal part of the binding site and a self-assembling domain containing a nanoparticle against PSA antibody with variable domain sequences. The PSA-specific variable structural domains of the nanobodies were selected from a library of phage genes immunized against dromedary camels and produced by heterologous expression in E. coli. After recrystallizing the investigator’s S-layer fusion protein on a gold chip precoated with sulfated SCWP, the single-molecule protein lattice was used as the sensing layer of a surface plasmon resonance biosensor to detect PSA. Lieven Huang and colleagues (Huang et al. 2005) chose a similar approach to prepare a new SPR biosensor. The researchers selected 24 nanobodies capable of targeting PSA and cAbPSA-N7 with higher affinity, and better kinetic parameters were selected to make the biosensor. It was shown that this biosensor could directly detect PSA concentrations down to 10 ng/ml, while this detection limit can be increased to sub-nanogram/ml PSA levels by introducing intercalation detection involving biotinylated secondary antibodies and gold nanoparticles modified with streptavidin. In recent studies, Xin Liu et al. (Liu et al. 2018) and Stefan Belicky et al. (Belicky et al. 2017) have prepared different biosensors that are prepared similarly by selecting one or more PSA-selective nanobodies covalently bound to a metal layer on SPR technology for PSA recognition. These biosensors above provide These biosensors offer the possibility of more accessible and more accurate observation of PSA changes and have the potential for clinical diagnosis.

Nanobody applications targeted PSMA

Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein expressed on the cell membrane. It has been shown that PSMA shows a specific high expression pattern in prostate cancer (PCa) and low expression in normal tissues (prostate, small intestine, salivary and lacrimal glands, and kidney), and its expression level is highly correlated with the aggressiveness of PCa (Wang et al. 2022). The extracellular structural domain of PSMA is large and easy to design target molecules. These properties have attracted the attention of many researchers, making PSMA a classical target for prostate cancer; for example, PSMA-centered PSMA-PET/CT has been considered the most sensitive and specific method for diagnosing recurrent prostate cancer (Afshar-Oromieh et al. 2019). Currently, most of the proteins that have been found to bind the extracellular region of PSMA with sufficiently high affinity (nanomolar range) are monoclonal antibodies or antibody fragments. This type of binding has certain shortcomings; the long serum half-life and wide biodistribution of antibodies tend to reduce the signal-to-noise ratio and maintain them in circulation for a long time. When antibodies are conjugated with cytotoxic radioisotopes, toxic effects may be increased. Therefore researchers further explored the application of nanobodies in the field of PSMA.

Back in 2012, Mehdi Evazalipour et al. designed relevant nanobodies against PSMA. In this study, the researchers immunized dromedary camels subcutaneously and intramuscularly using recombinant PSMA, LNCaP cells, and peptides containing 28 amino acids consistent with the outer surface structural domain of PSMA and isolated three camel IgG isoforms, including conventional (IgG1 160 kDa) and heavy chain (IgG2, IgG3 90 kDa) antibodies. The specificity and reactivity of the various IgG subtypes to PSMA were then determined by enzyme-linked immunosorbent assay (ELISA) and immunocytochemical studies for specific reactions with PSMA-expressing cell lines. The study's results confirmed that the camel species’ sera contained approximately 68% hcAb and that these hcAbs were typical for PSMA. Subsequently, they further confirmed in a 2014 publication that this VHH targeting for PSMA could be applied in molecular imaging of prostate cancer (Evazalipour et al. 2014). In this study, they similarly used ELISA and flow cytometry to assess the binding characteristics of nanobodies obtained by immunization with dromedary camels, followed by radiolabeling with (99 m) Tc in the hexameric histidine tail of selected nanobodies, and then assessed cell binding capacity and internalization on PSMA (positive) LNCaP and PSMA (negative) PC3 cell lines. In vivo tumor targeting in LNCaP and PC3 xenograft mice were analyzed by SPECT/MicroCT and tissue sampling. The results showed better targeting and higher internalization of PSMA30 among the screened VHH (Tc-99 m-labeled PSMA6 and PSMA30) both in vivo and in vitro. Although the above two studies do not appear to be complex today, they provide possible options to explore the clinical applications of nanobodies further.

In a 2015 study, Kristell and colleagues (Chatalic et al. 2015) designed a novel PSMA nanobody (JVZ-007) labeled with 111In targeting SPECT/CT imaging. The investigators immunized dromedary camels with four PCa cell lines (LNCaP, PC346C, VCaP, and MDA-PCa-2b) to obtain a library of nanobodies targeting PSMA. Nanobodies JVZ-007 were selected by phage biopanning to be evaluated as imaging probes. The C-terminal histidine tag of the nanobodies was subsequently replaced with a single cysteine to allow site-specific attachment of the radiolabeled chelates. JVZ-007-c-myc-his was then coupled to p-SCN-Bn-DTPA via lysine residues to allow radiolabeled 111 for SPECT imaging of PCa. JVZ-007-cys was site-specifically coupled to DTPA via reaction with maleimide-DTPA. The final analysis was performed by internalization of various PCa cell lines and patient-derived xenografts (PDX). The targeting properties of radiolabeled nanobodies were evaluated in vivo using nude mice bearing PSMA-positive PC-310 and PSMA-negative PC-3 tumors. The study showed that 111In-labeled anti-PSMA nanoantibodies, with good tumor targeting, low uptake in non-target tissues, and low renal retention, provided excellent SPECT-CT imaging of PCa within hours after injection. Compared to the study by Mehdi Evazalipour et al., Kristell's work further explores the superiority of anti-PSMA nanobodies compared to conventional tracers. We also speculate that the low renal retention of this nanobody may provide additional options for those older, really insufficiently functioning patients.

Since the work of Mehdi Evazalipour, and Kristell confirmed the feasibility and advantages of creating radiolabeled PSMA-targeting nanobodies over conventional tracers, we further wondered how effective the nanobodies would be compared to other new tracers, and Eline et al. PSMA-617, PSMA-I&T and the nanobody JVZ-007 (Ruigrok et al. 2021). The results showed that compared to the two small molecule inhibitors 177Lu-JVZ-007 may bind to different PSMA epitopes with very low binding levels on kidney and salivary gland tissues, making it a potential tracer, but the low tumor binding rate in vitro makes it unsuitable as a therapeutic tracer in its current form. Therefore, some investigators prepared multivalent nanobodies to improve the PSMA affinity of JVZ-007 (Yi-kai et al. 2022) and confirmed that bivalent nanobodies, which have higher yields and affinity levels than monovalent nanobodies and possess endocytosis efficiency no less than monovalent nanobodies, are important candidates for future development of PSMA-based therapeutic reagents for oncology.

Application of nanobodies in prostate cancer treatment

As mentioned earlier, with the increasing understanding of the process of cancer development, targeted therapy using the selectivity and specificity of antibodies can be more effective in reducing toxic side effects and has become one of the main modalities of cancer treatment at present (Maniam and Maniam 2021). The small volume of nanoantibodies can overcome the shortcomings such as poor permeability of traditional antibodies, and the use of nanoantibodies for targeted delivery of lethal substances and targeted radionuclide therapy has become a hot research direction, and this trend is also seen in the research of prostate cancer.

Recombinant immunotoxins

Immunotoxins (ITs) are a class of antibody-cytotoxin coupled chimeric molecules with specific cell killing ability. They target tumor cells by binding to tumor cell surface antigens or receptors through carrier molecules, inhibiting tumor cell protein synthesis or activating important apoptotic proteins to cause apoptosis, opening a new avenue for targeted therapy of malignant tumors.

Yutong Xing et al. (Xing et al. 2021) prepared a novel immunotoxin (JVM-PE24X7) using JVM and a modified Pseudomonas exotoxin A (PE) toxin (PE24X7), based on previous studies showing that the anti-PSMA nanoantibody JVM has ideal binding strength and selectivity for PSMA receptors, and tested its potential in prostate cancer treatment through a series of experiments. potential. Since the expression level of the PSMA receptor is important for the anti-tumor function of JVM-PE24X7, the researchers first identified PSMA-positive cell lines (LNCaP, C4-2B, and 22Rv1 cell lines), and PSMA-negative cell lines (PC-3 cells), respectively, by immunofluorescence staining and protein blot analysis assays. On this basis, the affinity of JVM-PE24X7 for PSMA was determined by ELISA, and the results showed that the Kd value of JVM-PE24X7 in PSMA-negative cell lines (> 1000) was much greater than its Kd value (24.4 nM) in PSMA-positive cell lines (LNCaP lines), confirming that JVM-PE24X7 has ideal high binding affinity and selectivity.

The immunotoxin based on targeting PSMA prepared by Yutong Xing et al. lacked an inhibitory effect on the PC-3 cell line. An interesting issue here is that PSMA is normally highly expressed in advanced prostate cancer, but PC-3 cells, which are hormone-resistant, are PSMA-negative cell lines; the reason for this is unknown and reminds us to develop new recombinant immunotoxins to remedy this aspect. Since vascular endothelial growth factor receptor 2 (VEGFR-2) is highly expressed on prostate cancer PC-3 cell lines, Samira Shajari designed a recombinant immunotoxin (VGRNb-DT) with truncated recombinant diphtheria toxin (DT) coupled to VEGFR-2 specific nanoantibodies (Shajari et al. 2022). The researchers evaluated the cytotoxicity of the immunotoxin by MTT assay on VEGFR-2-positive PC-3 cell lines, where overexpression of VEGFR-2 in PC-3 cell lines allowed the immunotoxin to recognize them by anti-VEGFR-2 nanoantibodies. The results showed that cell survival in PC-3 cells was significantly reduced at DT concentrations higher than those above 5 μg/ml, while different concentrations of DT were positively correlated with cell death in prostate cancer PC-3 cells.

Antibody–drug couples (ADCs)

Currently, ADCs are one of the most hotly researched anticancer drugs. ADCs are capable of targeted delivery of chemotherapy to tumor cells while preserving normal cells and can alleviate the major clinical hurdles of conventional chemotherapy, thus providing a wide range of therapeutic options. the main components of ADC drugs are antibodies, which should have the following characteristics (Khongorzul et al. 2020): (i) target specificity, i.e., the antibody should deliver cytotoxic drugs to tumor cells. (ii) Target binding affinity, i.e., the antibody should have a high binding affinity to tumor cell surface antigens. (iii) Antibodies should also have good retention, low immunogenicity, low cross-reactivity, and appropriate chain binding properties. The advantages of small size, high affinity, and high stability of nanobodies allow them to be considered as ideal antibodies for ADC drugs.

Ian Nessler and colleagues designed three nanoantibody drug couples to select the best framework for controlling internalization, clearance, and tissue penetration (Nessler et al. 2020). Each of the three targeted PSMA deemed couples has different properties, where the first construct (V_H2-V_H1-HLE) is a fusion between three V_H structural domains: two PSMA-binding structural domains with different epitopes (V_H1 and V_H2) are conjugated to an albumin-bound “half-life extender” (HLE) VH structural domain, yielding V_H2-V_H1-HLE. Since targeting multiple epitopes leads to antigen cross-linking and rapid internalization, binding albumin slows plasma clearance. Therefore, this coupling was designed for rapid internalization and slow clearance. The second construct (V_H1-HLE) consists of a monovalent PSMA-binding domain (V_H1) linked to an albumin-binding domain via a linker to form the V_H1-HLE construct. The lack of ability to cross-link the receptor slows internalization. A third construct (V_H2-V_H1) lacks the HLE structural domain to compare the effects of different clearance rates. All constructs were site-specifically linked to the alkylating agent DGN549 at the C-terminus for potency/efficacy studies. The results of the study showed that all three couples showed inhibition of PSMA-expressing prostate cancer in both in vitro and in vivo experiments.

More interestingly, in this study in vivo experiments showed that V_H1-HLE-DGN549 had the lowest cytotoxic potency of the three couples, yet in vivo experiments showed that it showed the greatest efficacy in all experimental groups, and tumor histological images and whole animal imaging showed that V_H1-HLE-DGN549 had the strongest tissue penetration. The investigators believe the reason for this result is that in vivo, the extravasation of ADCs drugs from the bloodstream is the rate-limiting step and determines the total uptake of the drug by the tumor. Therefore, faster internalization did not deliver more total payload to the tumor. Fast internalization instead limited the penetration distance of V_H2-V_H1-AF680 and V_H2-V_H1-HLE-AF680 in tumor tissues, and thus they reached fewer cells. Although V_H1-HLE-AF680 and V_H2-V_H1-HLE-AF680 deliver the same amount to tumors, the slower internalizing V_H1-HLE-AF680 can penetrate farther into the tumor to target and kill more cells.

ADCs drugs are known as “magic missiles”, which combine the high selectivity of antibodies with the high efficiency of antitumor drugs in cytotoxicity, and are widely studied in solid tumors such as lung cancer (Ricciuti et al. 2021). Meanwhile, an increasing number of investigators have explored the use of ADCs drugs in advanced prostate cancer, A phase II clinical study included patients (N = 119) who progressed to metastatic chemoresistant prostate cancer after receiving abiraterone or enzalutamide receiving PSMA antibody–drug coupling (PSMA ADC) 2.5 or 2.3 mg/kg IV q3w for up to 8 cycles. The results showed that PSA decreased by l50% in 35% of patients; CTC counts decreased by 50% or more in 78% of patients (Petrylak et al. 2020). Also, we see that the study by Ian Nessler et al. confirms the promise of nanobody ADCs targeting PSMA, Although their study is still preclinical, it provides further insight into the internalization of ADCs drugs in solid tumors, screening for more stable drug patterns, and opening up possibilities for subsequent clinics.

Summary and outlook

In the face of the progressively higher epidemiological profile of prostate cancer, early screening is undoubtedly the most important management tool. Current prostate cancer screening based on PSA testing may cause false positive screening, and in large randomized controlled studies (Epstein et al. 2016), about 1/4 to 1/3 of men who underwent PSA screening had at least 1 positive screening result, resulting in a transitional diagnosis of prostate cancer. Therefore, researchers are constantly searching for new and effective screening tools, and some of the studies for PSA and PSMA mentioned in this paper provide new diagnostic options. The advantages of high stability and specificity of nanoantibodies are the basis of all research. The development of new nanoantibody biosensors targeting PSA and PSMA may be a new direction for future diagnostic development.

It is well known that androgen deprivation therapy is an important cornerstone of prostate cancer and has led to a dramatic decrease in associated mortality since 1994 and is widely used in all phases of prostate treatment (Chaput and Sumar 2022). However, the current androgen deprivation-centered treatment paradigm has never been able to address the fact that patients will eventually and irreversibly enter the destructive resistance phase. Therefore, investigators continue to explore new therapeutic approaches for prostate cancer. Still, ICI has failed to make a breakthrough in prostate cancer at a time when immune checkpoint inhibitor therapy is in full swing. The OS results of the phase III IMbassador 250 study (Powles et al. 2022) showed that atezolizumab combined with enzalutamide did not provide a survival benefit in patients with metastatic debulk-resistant prostate cancer (mCRPC). Because of the extremely low likelihood of the trial meeting its primary endpoint, continuation was not considered in the best interest of patients, and the study was stopped early after a planned Independent Data Monitoring Committee (IDMC) meeting to assess safety. The “coldness” of immunotherapy in prostate cancer is likely related to the mutational load of prostate cancer and the low number of infiltrating lymphocytes, and the need to break through the limitations to make immunotherapy useful is a major concern. Among the studies mentioned in this paper, recombinant immunotoxins and ADC drugs are breakthroughs. Targeting PSMA recombinant immunotoxins have the advantage of high affinity and low concentration onset. In contrast, ADC drugs have the benefit of high penetration, which lays the foundation for further exploring new immunotherapies. We expect these results to be translated into the clinic.

Unlike traditional mAb, nanobodies have a small size, high antigen specificity, high affinity, and stability, and can target TAA, tumor microenvironment, or immune cells and bind to hidden antigenic epitopes. At present, about nanobody antitumor drugs are still in the beginning stage, and the related research mentioned in this paper is still in the preclinical stage. Still, we have reasons to believe that with the further application and development of bioengineering technology, nanobodies are expected to replace traditional mAb and may bring a breakthrough in the treatment of prostate cancer.

Author contributions

All authors contributed to the study conception and design. This article was co-authored by JS and XL. They are co-first authors; SG and JZ collected the relevant data; XW created the graphs needed for the article; XL reviewed the full text and is the corresponding author of this article.

Funding

This project was supported by the Tianjin Science and Technology Program (Tianjin Natural Science Foundation Project), 19JCZDJC37000.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

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