Antibody-Based Therapeutics for the Treatment of Renal Cell Carcinoma: Challenges and Opportunities

Abstract

Renal cell carcinoma (RCC) is among the top 10 most common cancers in both men and women with an estimated 75 000 cases each year in the US. Over the last decade, the therapeutic landscape for patients with metastatic RCC has significantly evolved, with immunotherapy emerging as the new front-line therapy. Despite significant improvement in toxicity profile and survival outcomes, key concerns such as patient selection, treatment sequencing, and intrinsic and acquired resistance remain unresolved. Emerging options such as antibody-based therapeutics (eg, anti-CD70, anti-CA9, and anti-ENPP3) are being explored in clinical trials for patients with cancer resistant or refractory to current immunotherapies. Despite positive results for hematological cancers, breast cancer, and more recently bladder cancer, most antibody-based therapies failed to improve the outcomes in patients with advanced RCC. This underscores the need to understand the underlying causes of failed responses to this treatment class, which will ultimately support the rational design of more effective and tolerable treatments. In this review, we summarize the evolving landscape of RCC therapeutics and describe recent clinical trials with emerging antibody-based therapeutics. We also describe the challenges that need to be overcome for the successful creation of therapeutic antibodies for treating RCC.

Despite positive results for other cancers, most antibody-based therapies failed to improve outcomes in patients with advanced renal cell carcinoma (RCC). This review summarizes the evolving landscape of RCC therapeutics and describes recent clinical trials with emerging antibody-based therapeutics, as well as the challenges to be overcome for the successful creation of therapeutic antibodies for treating RCC.

Implications for Practice.

In this review, the reader will receive a thorough overview of the various antibody-based therapeutic options for patients with advanced renal cell carcinoma. As the molecular biology and cytogenetics of RCC being explored more, various targeted medications have been investigated to improve patient outcomes; additionally, they provide physicians the chance to tailor therapies for specific patients. In our review, we highlighted the limitations of previous and most recently conducted clinical trials on antibody-based therapeutics and the approaches put forth to overcome those issues.

Introduction

Renal cell carcinoma (RCC) is the most frequent subtype of kidney cancer with an estimated 400 000 occurrences globally.¹ Overall, the lifetime risk for developing kidney cancer in men is approximately 1 in 46 (2.02%), and for women is approximately 1 in 80 (1.03%).² Nearly 30% of patients present with de novo metastatic disease and almost 30% of those with initial localized disease will eventually develop metastases after surgical resection of the primary tumor.³

Over the past 2 decades, there have been significant changes in the management and treatment of metastatic RCC.⁴ First-generation immunotherapy with interleukins or interferon which was previously standard of care is now fallen out of favor due to subpar results and high toxicity.^5,6 Subsequently, the prognosis for both progression-free survival (PFS) and overall survival (OS) was significantly improved by the introduction of tyrosine kinase inhibitors (TKI), primarily VEGF receptor inhibitors.⁷ Later the immune checkpoint inhibitors (ICI) emerged which alone or in combination with TKIs have led to better oncologic outcomes.^8,9 Because RCC is considered as an immunogenic tumor with high numbers of immune cells, targeted immunotherapy is a best alternative to antiangiogenics.^10-12 Currently, multiple therapies are available for patients with de novo metastatic or recurrent RCC, owing to collaborative efforts led by scientists and clinicians. Despite improved response rates to currently available therapies, there is still a large fraction of patients (~50%-70%) who develop treatment resistance and disease progression. Therefore, there is an unmet need to develop new therapeutic options for these patients.⁷ In our review, we will discuss a new class of therapeutics, namely antibody-based therapeutics, that has been tested in clinical trials.

Materials and Methods

We used MEDLINE to retrieve pertinent literature related to pivotal trials and presentations from the American Society of Clinical Oncology (ASCO) and the European Society for Medical Oncology (ESMO) conferences. We used the keywords “renal cell carcinoma,” “RCC,” “metastatic RCC,” “advanced RCC,” “antibody-based therapies,” “immunotherapy,” “clinical trials,” and “emerging drugs,” and restricted our search to articles published since 2010.

The Evolving Therapeutic Landscape of Metastatic RCC

2005-2015: The “VEGF Inhibitors” Era

Before 2005, first-generation immunotherapy using cytokines such as IL-2 or IFN-γ was the gold standard for the treatment of metastatic RCC, but was associated with severe toxicities and poor response rates.^5,6 The discovery of VHL inactivation in RCC and its impact on tumor angiogenesis led to the clinical trials evaluating the efficacy of tyrosine kinase inhibitors (small-molecule inhibitors), particularly those targeting the VEGF receptor.^13,14 In metastatic RCC, VEGF inhibitors such as sorafenib, sunitinib, and pazopanib led to an ~30% response rate and overall survival (OS) benefit over first-generation immunotherapy.⁷ Similarly, genetic alterations of the mTOR signaling pathway frequently observed in RCC have led to phase III trials demonstrating the efficacy of mTOR inhibitors temsirolimus and everolimus in a subset of patients with advanced RCC.¹⁵ The treatment paradigm then further evolved with the advent of immune checkpoint inhibitors, which were initially used for treating other aggressive cancers such as melanoma and lung cancer.^16,17

2015-Present: The “Immune Checkpoint Inhibitors” Era

Immune checkpoint inhibitors were used for the treatment of mRCC, given immunogenicity and the high number of infiltrating lymphocytes in these tumors (TIL).^11,12,18 Since FDA approval of nivolumab as a second-line therapy for mRCC in 2015, immunotherapy has progressed to become a key treatment for mRCC.¹⁹ Following numerous phase III randomized trials, 4 treatments including immune checkpoint inhibitors (Nivolumab, Ipilimumab, Pembrolizumab, and Avelumab) have shown improved survival benefits compared to the tyrosine kinase inhibitor Sunitinib alone and constitute the new backbone for treating advanced RCC. One approach consists of a combination of 2 immune checkpoint inhibitors, Nivolumab (anti-PD1) and Ipilimumab (anti-CTLA4) which was approved in 2018.²⁰ The other treatments include one immune checkpoint inhibitor in combination with a tyrosine kinase inhibitor (Table 1; Fig. 1).

Table 1.

Comparison of the phase III clinical trials investigating immune checkpoint inhibitors.

Phase III trials	CheckMate 21421	Keynote 42622	CheckMate 9ER23	Javelin Renal 10124	CLEAR25	Immotion 15126	COSMIC-31327
Treatment	Nivo + Ipi vs. Sun	Pembro + Axi vs. Sun	Nivo + Cabo vs. Sun	Ave + Axi vs. Sun	Lenva+Pemb vs. Lenva+Ever vs. Sun	Atezo +Bevaci vs. Sun	Cabo + Nivo +Ipi vs. Nivo + Ipi
Number of patients	1096	861	651	886	1069	915	855
Median follow-up (months)	32	30.6	18.1	13	24	24	20.2
Median OS (%)	30Mon: 60 vs. 47	12Mon: 90 vs. 78	12Mon: 85.7 vs. 75.6	12Mon: 86 vs. 83	24Mon: 79.2 vs. 66.1 vs. 70.4	24Mon: 63 vs. 60	–
Median PFS (months)	11.6 vs. 8.4	15.1 vs. 11.1	16.6 vs. 8.3	13.8 vs. 7.2	23.9 vs. 14.7 vs. 9.	11.2 vs. 7.7	NR vs. 11.3
ORR (%)	42 vs. 34	60 vs. 40	55.7 vs. 27.1	55.2 vs. 25.5	71vs. 53.5vs. 36.1	37 vs. 33	43 vs. 36
CR (%)	11 vs. 2	9 vs. 3	8 vs. 4.6	3.4 vs. 1.8	16.1vs. 9.8vs. 4.2	5 vs. 2	3 vs. 3
PR (%)	31 vs. 32	51 vs. 37	47.7 vs. 22.6	48.6 vs. 25.2	54.9vs.43.7vs. 31.9	31 vs. 31	41 vs. 32
SD (%)	30 vs. 41	23 vs. 35	32.2 vs. 42.1	28.3 vs. 43.7	19.2 vs. 33.6vs. 38.1	39 vs. 39	43 vs. 36
PD (%)	22 vs. 16	11 vs. 17	5.6 vs. 13.7	12.4 vs. 19.4	5.4 vs. 7.3 vs. 14	18 vs. 19	8 vs. 20
Adverse effects (grade≥3)	76 vs. 71	58 vs. 55	75.3 vs. 70.6	71.2 vs. 71.5	82.4vs. 83.1vs. 71.8	40 vs. 54	73 vs. 41
Cost-effectiveness	Nivo + Ipi28	Pembro + Axi29	Sun23	Sun30	–	–	–

Abbreviations: OS, overall survival; PFS, progressive free survival; ORR, objective response rate; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; Nivo, nivolumab; Ipi, ipilimumab; Sun, sunitinib; Pembro, pembrolizumab; Axi, axitinib; Cabo, cabozantinib; Ave, avelumab; Lenva, lenvatinib; Ever, everolimus; Atezo, atezolizumab; Bevaci, bevacizumab.

Figure 1.

Forest plots showing the association of investigational therapies with risk of mortality and progression-free survival in advanced RCC.

However, the choice of first-line systemic treatment remains challenging as no predictive biomarker to guide treatment selection has been approved to date.³¹ Patients’ comorbidities and treatment side effects should be considered on a case-by-case basis. Despite improved objective response rates (~40%-70%) with current immunotherapy regimens, the majority of patients develop treatment resistance and disease progression.^32,33 Therefore, there is a need to develop novel treatments for patients with refractory or recurrent RCC.³

Emerging Therapeutics for Advanced Renal Cell Carcinoma

Small-Molecule Inhibitors

The growing understanding of RCC biology and progress in bioengineering has enabled the design of novel targeted therapies. This includes small-molecule inhibitors and antibody-based therapies. With the increasing understanding of the VHL/HIF biology in RCC, inhibitors of the HIF-2 transcription factor represent an important line of investigation.^34-37 Belzutifan is the first HIF-2 inhibitor to be evaluated in phase III trials for the treatment of advanced ccRCC. It has recently become the first-ever HIF-2 inhibitor approved by the FDA for the treatment of VHL-associated tumors but not yet for non-hereditary renal cell carcinoma.³⁸

Overcoming immunosuppressive barriers within the tumor microenvironment (TME) has become another approach.³² A small-molecule against the adenosine 2A receptor (A2AR) has shown promising antitumor activity in a first-in-human study.³⁹ Adenosine is a nucleoside that can be generated from extracellular ATP through the enzymatic activity of ectonucleotidases such as CD39, ENPP3, and CD73.⁴⁰ Adenosine binds to A2AR inhibitory receptors expressed by T and NK cells, resulting in immunosuppression. Compared to other solid tumor histology, the adenosine pathway genes ADORA2A (A2AR), ENPP3, and NT5E (CD73) are substantially expressed in RCC.⁴¹ Intertumoral hypoxia may also contribute to the formation of extracellular adenosine in RCC tumors by increasing the expression of CD39 and CD73 and encouraging the release of intracellular ATP.⁴² The phase I trial with the A2AR antagonist CPI-444 (Ciforadenant) included 68 patients with RCC with more than 72% of them having previously unresponsive or refractory anti–PD-1/PD-L1 antibody therapy.⁴³ Ciforadenant was associated with increased recruitment of CD8+ T cells in tumors, diversification of the T-cell receptor (TCR) repertoire in circulating T cells, and durable clinical benefit with a median progression-free survival of 4.1 months and an estimated OS of 69% at 16 months. Therefore, A2AR antagonists represent a promising new form of immune checkpoint inhibitors distinct from PD-1/PD-L1.³⁹ Furthermore, a recent phase II trial was conducted on 69 patients to evaluate the effectiveness of the combination of the mTOR inhibitor (Everolimus) and the oral glutaminase inhibitor (Telaglenastat) versus Everolimus and placebo. Glutaminase is considered a key enzyme in the glutamine-dependent biosynthesis of tumor metabolic intermediates. The combination of Telaglenastat and Everolimus induces synergistic anti-tumor effects and improves progression-free survival (PFS), with a median PFS of 3.8 months compared with 1.9 months in the Everolimus and placebo group.⁴⁴

Antibody-Based Therapeutics

Antibody-based therapeutics are also emerging as future tools for treating advanced RCC and can be categorized into 2 groups. The first category includes “naked” humanized antibodies which are used directly for therapy. For instance, immune checkpoint inhibitors are monoclonal antibodies that prevent the interaction of immuno-inhibitory ligands with their receptors expressed on the surface of immune cells.⁹ Some monoclonal antibodies can also induce antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) naturally. In both mechanisms, natural killer (NK) cells or macrophages recognize the Fc portion of antibodies on the surface of tumor cells leading to tumor cell lysis or phagocytosis respectively.^45-47 The second group includes engineered antibodies such as antibody-drug conjugates, radionuclide-conjugated antibodies, bi- and tri-specific antibodies, functionalized liposomes, and chimeric antigen receptor T cells (CAR-T).^48-51

The first-ever monoclonal antibody OKT3 (anti-CD3) was approved by the FDA in 1986. Since then, more than 79 therapeutic antibodies have been approved for clinical use, representing a global market of US$115.2 billion in 2018.⁴³ In the following section, we will describe results from recent clinical trials evaluating new antibody-based therapeutics in the treatment of RCC (Fig. 2).

Figure 2.

Recent clinical trials evaluating new antibody-based therapeutics in the treatment of RCC.

Abbreviations: CA9, carbonic anhydrase 9; CAR-T, chimeric antigen receptor T cells; TIM-1, T cell immunoglobulin mucin-1; ADC, antibody-drug conjugate; ENPP3, ectonucleotide pyrophosphatase/phosphodiesterase 3; PFS, progressive-free survival; OS, overall survival.

Antibody-Based Therapeutics in Metastatic RCC: Early Lessons From the Clinic

Anti-CA9

Carbonic anhydrase IX (CA9), a membrane protein that promotes cell proliferation and angiogenesis in response to hypoxia.⁵² CA9 is overexpressed in different cancers, including RCC, and is activated by hypoxia-inducible factor 1 (HIF1). It is found in more than 80% of primary and metastatic RCC and more than 95% of clear cell RCC.⁵³ As a result, CA9 is a powerful biomarker of RCC and a possible target for RCC immunotherapy.⁵⁴

Chimeric antigen receptor T cells (CAR-T) models and monoclonal antibodies (mAb) targeting CA9-expressing cells have been recently investigated in clinical trials. CAR-T models are created by inserting the gene for a chimeric T-cell receptor-antibody targeting a specific cancer marker into T cells, enabling the modified cells to target and eliminate cancerous cells.⁵⁵ In a phase I/II study, 12 patients with metastatic RCC were injected with CAR-T targeting CA9.⁵⁶ Following infusion, patients generated anti-CAR-T antibodies and anti-CAR-T immune responses. Furthermore, CAR-T infusions resulted in grades 2-4 liver enzyme abnormalities, necessitating therapy discontinuation in 4 out of 12 patients. T-cell infiltration surrounding bile ducts and CA9 expression on bile duct epithelium were found in liver biopsies supporting off-tumor on-target toxicity. The trial was discontinued before achieving therapeutic or maximum tolerated dosage due to the introduction of competitive drugs into the field and the presence of high toxicity.⁵⁶

Few phases I/II clinical trials have evaluated Girentuximab, a chimeric monoclonal antibody that binds selectively to CA9 and induces ADCC. In the initial safety study, no grade 3 and 4 toxicity was observed and antitumor activity was limited to one complete response out of 36 patients.⁵⁷ To improve the antitumor activity of Girentuximab in advanced renal cell carcinoma, a second trial tested anti-CA9 antibodies with a low dose of interleukin IL-2.⁵⁸ Eight patients out of 35 (23%) have shown clinical benefit, which includes 3 patients with partial response and 5 patients with stable disease. Median OS was 22 months. Girentuximab was also tested in a randomized, double-blind, placebo-controlled phase III clinical study as an adjuvant treatment in 864 individuals presenting with RCC and a high risk of postoperative recurrence. However, there was no difference in disease-free survival (DFS) or OS between the 2 groups of patients.⁵⁹

An alternative therapeutic strategy recently investigated is the use of Girentuximab conjugated with Lutetium-177, a radioligand that delivers radiation to the surrounding environment. Lutetium-177 has recently been found to be useful for treating metastatic castration-refractory prostate cancer.⁶⁰ In RCC, a phase I trial reported data on 23 patients with metastatic disease treated with ¹⁷⁷Lu-labelled Girentuximab and stable disease was observed in 74% of patients.⁶¹ In a second trial on 14 patients, most of them developed severe and prolonged grades 3-4 thrombocytopenia which led to treatment discontinuation and only 1 patient demonstrated significant antitumor activity according to Response Evaluation Criteria in Solid Tumors (RECIST) criteria.⁶² Altogether, these clinical trials indicate that despite CA9 being considered as the most promising tumor target, CAR-T, and monoclonal antibodies against CA9 have failed to demonstrate clinical benefit in patients with mRCC. CA9 is highly expressed in RCC, but normal tissue expression can lead to serious toxicity making it a less clinically useful target. In addition, a recent multi-center phase III trial on 252 mRCC investigated the use of conjugated Girentuximab with Zirconium (TLX250-CDx/ 89Zr-DFO-girentuximab) as a radiolabeled isotope to diagnose clear cell RCC (ccRCC) using the PET scan. The initial results are promising with an 86% sensitivity, 87% specificity, and 93% positive predictive value. All of this indicates that TLX250-CDx has the potential to become a new clinical standard in the diagnosis of ccRCC as a non-invasive diagnostic tool (NCT03849118).

Anti-TIM1

CDX-014 is an antibody-drug conjugate (ADC) that targets T-cell Immunoglobulin Mucin-1 (TIM-1), also known as Kidney Injury Molecule 1 (KIM-1), a transmembrane glycoprotein that is highly expressed in tubular cells in response to ischemia.⁶³ It is found to be highly expressed in 70% of RCC, especially in clear cell and papillary subtypes.^64,65 A phase I first-in-human study was conducted on 16 individuals.⁵⁴ CDX-014 demonstrated anticancer efficacy, resulting in sustained partial response and stable disease in 31% of patients. The 2 patients who had the most significant tumor shrinkage had strong TIM-1 expression on tumor biopsies. Adverse events of grade 3 or higher included hyperglycemia (19%), urosepsis (6%), and one multiple organ failure (6%), which resulted in one treatment-related death. Overall, despite some early evidence of efficacy, this ADC has limited clinical use due to high toxicity profile.^66-68

Anti-CD70

CD70, also known as CD27L or TNFSF7, is a type II integral membrane protein found on activated T cells, B cells, and mature dendritic cells.⁶⁹ Its receptor CD27 is expressed on a high proportion of NK cells as well as resting T and B cells. Signaling through CD27 promotes T-cell and B-cell activation upon CD27L binding.^70,71 CD70 is overexpressed in several solid tumors and hematologic malignancies.^72-74 Although the causes are unknown, this ligand has characteristics consistent with those of a tumor-associated antigen due to its limited expression in healthy but preferential representation in malignant tissue.^73,75 In renal cell carcinoma, CD70 has been found to be overexpressed in a vast majority of cases compared to normal kidney tissue.^73,76,77 Importantly, CD70 is rapidly internalized following antibody attachment, making it a viable target for antibody-based cytotoxic drug delivery.^75,78

AMG 172 is an ADC constituted of a humanized IgG1 monoclonal antibody (mAb) against CD70 conjugated with a microtubule inhibitor (maytansine).⁷⁹ It was investigated in a phase I trial of 37 patients with refractory or relapsed RCC. Dose-limiting toxicity that prompted therapy cessation included thrombocytopenia and hepatic damage. Out of 37 patients, 21 reached the maximum tolerated dose and the response was: 2 patients (5.4%) had a partial response, 6 patients (16.2%) had stable disease, and 13 patients (35.1%) had progressive disease. Based on the limited antitumor activity and treatment-related toxicities, no further trials are expected.⁷⁹

A second ADC, called SGN-CD70A, was also evaluated in a phase I human study.⁸⁰ SGN-CD70A is an anti-CD70 antibody conjugated with a pyrrolobenzodiazepine which induces DNA breaks and apoptosis. In total, 18 patients with metastatic RCC were enrolled and most patients discontinued treatment because of progressive disease (56%) or adverse events (28%). Like AMG 172, no further trials are ongoing using SGN-CD70A. Further work is needed to understand the causes of treatment-related toxicities and limited antitumor activity.

Anti-ENPP3

Ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) is one of the most overexpressed surface proteins in RCC, particularly on clear cell histology with up to 94% expression.^81,82 In contrast, ENPP3 is found weakly expressed in a fraction of renal tubules and activated basophils and mast cells.⁸³ AGS-16C3F is an ADC against ENPP3 attached to a microtubule inhibitor. Following successful pharmacokinetics and safety study, a phase II trial comparing AGS-16C3F to Axitinib was conducted in the previously 133 treated metastatic RCC.⁸⁴ Patients were randomized 1:1 and 63% of patients had completed the study at data cutoff. AGS-16C3F did not enhance PFS compared with Axitinib (2.9 months vs. 5.7 months, HR 1.676, P = .015). Similar observations were made for OS (13.1 months for AGS-16C3F versus 15.4 months for Axitinib; HR, 1.079; 95% CI, 0.681-1.707; P = .747). Based on the study findings, no further development of AGS-16C3F in mRCC will be undertaken.⁸⁴

Anti-CDH6

CDH6 is a cell-adhesion molecule of the cadherin family.⁸⁵ Protein expression was evaluated in a small subset of primary RCC specimens (39 patients) and CDH6 staining intensity was heterogeneous across samples. In non-human primates, dose-dependent toxicities of HKT288, an ADC against CDH6 carrying a microtubule inhibitor, were observed in corneal epithelium and skin but do not reflect on-target toxicity.⁸⁵ Data from the first-in-human study in 9 patients with advanced solid tumors was recently reported, including 5 patients with RCC. Three patients developed unexpected grade 2 neurological adverse events (1 seizure, 1 aphasia, and 1 encephalopathy). Further studies investigating the mechanisms of toxicity are critical before further trials with CDH6-targeting agents are pursued.

Current Challenges and Opportunities for Antibody-Based Therapeutics

So far, the therapeutic strategy has been mostly over-simplistic with the idea that a protein highly expressed in RCC is probably the perfect target for antibody-based therapeutics. However, our growing understanding of the cellular and molecular mechanisms of drug resistance and the complex nature of the tumor microenvironment provide important insights to explain the lack of success in recent clinical trials with therapeutic antibodies. In the following section, we describe the biological factors that influence the efficacy of antibody-based therapeutics and propose several strategies to improve the design of more effective and tolerable anticancer drugs (Fig. 3).

Figure 3.

Challenges and opportunities with antibody-based therapeutics.

On-Target Off-Tumor Cytotoxicity

This consists of recognition and destruction of normal tissues that have the shared expression of the targeted protein. The ideal tumor target would be a surface protein only expressed by tumors but, despite tumors accumulating mutations over time, only a few tumor-specific surface proteins have been identified so far (eg, EGFRvIII, mutant FGFR3).^86,87 However, overexpression of surface proteins is common in tumors and the expression ratio between normal and tumor tissue can guide the development of high-affinity antibody-based therapeutics. Recent clinical trials with a CAR-T directed against CA9 in RCC illustrate the on-target off-tumor cytotoxicity mechanism.^88,89 This highlights the crucial need to establish a full evaluation of target expression in normal human tissues before initiating in-human trials. With the recent advances in profiling technologies, several publicly available databases have been established to interrogate gene and protein expression in normal and tumor tissues (eg, GTEx, TCGA).⁹⁰ Recently, researchers have developed an interactive CAR clinical trial database combining gene and protein data to show the current “targetable landscape.”⁹¹ The GTEx consortium has recently released protein expression profiles of 12 000 genes across 32 normal human tissues using advanced mass spectrometry.⁹² These resources are helpful to interrogate the normal expression of druggable surface proteins (surfaceome) and anticipate potential on-target off-tumor cytotoxicity.⁹³ Such initiative will significantly drive the discovery of novel targets for next-generation immunotherapy.⁹⁴

Tumor Uptake

Another challenge to overcome in antibody-based therapies is ensuring that the antibody reaches the intended target. Solid tumors are characterized by a disorganized vasculature and a dense extracellular matrix that limits the tissue penetration of therapeutic antibodies. One approach is to produce antibody fragments with smaller sizes and higher diffusion rates.^95,96 There is poor blood flow and hypoxia because the blood vessels within solid tumors are largely immature and disordered.^97,98 In addition, plasma leakage into the interstitial space is caused by structural defects in solid tumor blood vessels and an overexpression of pro-angiogenic factors that raise the permeability of the vasculature.⁹⁸ High interstitial oncotic pressure can constrain the convective flow of therapeutic antibodies from the blood into the interstitial fluid of tumors and can cause tumor blood vessels to collapse.⁹⁹ One method to improve tumor uptake of therapeutic antibodies is to use pharmacological agents to modify tumor vasculature and tissue permeability.⁹⁶ Tumor absorption of therapeutic antibodies increased between 40% and 200% when given concomitantly with the vasculature-promoting drugs such as angiotensin II, tumor necrosis factor-alpha, interferon, and interleukin 2.^100-103

Tumor Heterogeneity

Intratumoral heterogeneity consists of the co-existence of several tumor clones within the same patient’s tumor, and it can be categorized as spatial heterogeneity or temporal heterogeneity.¹⁰⁴ Intratumoral heterogeneity has been well documented in RCC.^105-107 Intertumoral heterogeneity defines genotypic and phenotypic differences between different tumors within the same patients (ie, primary tumor versus metastasis). It can be attributed to multiple factors including somatic mutations, environmental pressures, tumor-immune interactions, and prior systemic therapies.¹⁰⁴ Intertumoral heterogeneity can be a major cause of resistance to therapy in metastatic cancer patients as patient selection for antibody-based therapeutics is usually based on target protein expression in resected primary tumor specimens. However, throughout the past few years, several studies have demonstrated molecular differences between primary RCC tumors and patient-matched metastases.^108-111 One group showed a discordance rate of 27% and 22% for PD-1 and PD-L1, respectively between patient-matched primary and metastatic ccRCC.¹¹² Importantly, both proteins were less expressed in metastatic lesions compared to primary tumors. One important caveat in clinical trials evaluating therapeutic antibodies for the treatment of RCC is the lack of information on protein expression in metastatic specimens. In the failed trial with SGN-CD70A, the authors acknowledged that it is uncertain if CD70 expression differed across primary and metastatic locations, or whether CD70 expression changed throughout therapy.⁸⁰ For CA9, one study has shown a very poor concordance rate (<50%) in metastatic lesions within the same patient.¹¹³ Altogether, this data highlights the need to select patients based on target protein expression in metastatic lesions. While obtaining metastatic biopsy samples can be challenging due to their invasiveness, a promising way to overcome this is through utilizing liquid biopsies.^114,115 In the context of antibody-based therapeutics, circulating tumor cells (CTCs) and extracellular vesicles (EVs) hold significant potential as they carry surface proteins expressed by the tumor they originate from. CTCs are disseminating cells from a primary tumor and are thought to form metastases at remote sites. Patients with metastatic disease usually have a higher CTC blood count than those with localized disease.¹¹⁶ In the blood of RCC patients, CA9 has been detected on the surface of CTCs, which provides a proof of feasibility for isolation of CTCs from patient blood to assess the expression of targets in metastasis-derived cells.¹¹⁷

Extracellular vesicles (EVs) are small particles (~30 nm to 5 µm) released naturally by all cell types including tumor cells. They contain molecular cargo (DNA, RNA, proteins) from donor cells and their detection in blood and urine offers new opportunities for cancer diagnosis and early detection of disease recurrence. Organ- or cancer-specific EVs can be detected directly from biofluids using cutting-edge technologies such as nanoscale flow cytometry and interferometric imaging.¹¹⁸ Several proteins upregulated in RCC have been found in urine-derived EVs, including ENPP3, PODXL, and AQP1.¹¹⁹ Other groups have reported the presence of CA9 and CD70 in blood- and urine-derived EVs. isolated from RCC patients.^120-122 These findings support the potential of liquid biopsies to guide treatment decisions in biomarker-driven clinical trials. However, CTC and EV research is still in an exploratory phase and further studies are needed to fully characterize the molecular profile of RCC-derived CTCs and EVs. and assess their clinical value for patient selection and predictive markers of treatment response.

Another interesting approach to determining target protein expression in inaccessible metastatic lesions is positron emission tomography (PET). Radiotracers can be conjugated into antibodies or ligands specific to tumors. Several radiotracers are currently under investigation, including 89Zr-Geruntuximab (anti-CA9) and 89Zr-Atezolizumab (anti-PD-L1).^123,124 A clinical study on 195 patients showed a sensitivity and a specificity of 86.2% and 85.9% for the detection of malignant renal masses with 89Zr-Geruntuximab.¹²⁵

Circulating Molecular Decoys

The primary target for the therapeutic antibodies is a protein expressed on the surface of tumor cells. However, alternative forms of the protein target can be found in circulation in the form of soluble protein or on the surface of extracellular vesicles. As described above, tumor cells release EVs as an intrinsic mechanism of cargo elimination and the EV proteome reflects the cell of origin.¹²⁶ Within EVs, surface proteins can preserve their structural conformation and topology. Interestingly, engineered EVs with specific cytokine receptors have been developed to reduce systemic inflammation by capturing pro-inflammatory cytokines IL-6 and TNF-a.¹²⁷ Similarly, circulating EVs expressing the virus entry receptor ACE2 were effective to neutralize SARS-Cov2 and inhibit its infectivity.^128-130 While EVs can be used as a therapeutic tool, they can also serve as a mechanism of resistance to antibody-based therapies. SARS-CoV-2 infected cells can release EVs carrying the virus spike protein and act as a decoy target for convalescent patient serum-derived neutralizing antibodies.¹³¹ Therefore, we can assume that circulating EVs can capture monoclonal therapeutic antibodies and prevent their accumulation in the target site (ie, metastatic lesions).^132-134 In line with this, a recent study has demonstrated the predictive value of circulating PD-1⁺-EVs in melanoma patients treated with nivolumab (anti-PD-1).¹³⁵ PD-1⁺-EVs can bind to nivolumab and higher levels of PD-1⁺-EVs were found in non-responder patients, which supports the hypothesis that EVs can act as molecular decoys to antibodies resulting in therapeutic resistance. Soluble proteins can be produced through proteolytic cleavage or released upon cell death. By being functional, they can also contribute to resistance to therapeutic antibodies through mechanisms similar to EVs. To date, there is no FDA-approved clinical intervention to inhibit EV biogenesis. Recently, it has been demonstrated the feasibility of eliminating soluble PD-L1 and PD-L1-positive EVs from patient’s blood by therapeutic plasma exchange (TPE).¹³⁶ During TPE the blood is passed through an apheresis machine separating plasma from cellular components. After one single treatment, ~70% of soluble PD-L1 and PD-L1-positive EVs were eliminated from the patient’s blood. A phase I trial is currently ongoing to investigate the therapeutic benefit of plasma exchange in melanoma patients treated with immunotherapy (NCT04581382).

Conclusions

Over the last decade, the therapeutic landscape for patients with metastatic RCC has greatly evolved, with tailored immunotherapy emerging as a new pillar in front-line treatment. Despite these major advances, critical issues like optimal patient selection, treatment sequencing, and overcoming intrinsic and acquired resistance remain to be solved. Antibody-based therapeutics have shown promise in other malignancies, but the lack of success in recent clinical trials in kidney cancer underscores the need to better understand resistance mechanisms and other biological barriers to their efficient use. Importantly, a systematic evaluation of the expression and regulation of targetable proteins in tumors and normal tissue is essential to anticipate treatment-related toxicities and to develop new drugs.

Funding

This research received no external funding.

Conflict of Interest

The authors indicated no financial relationships.

Author Contributions

Conception and design: F.L., B.A.C., J.C. Collection of data: A.M.M., J.O., I.G. Manuscript writing: A.M.M., R.N., R.R.P., I.G., J.O., B.A.C., F.L. Final approval of the manuscript: All authors. Accountable for all aspects of the work: All authors.

Data Availability

No new data were generated or analyzed in support of this research.