**Influence of Vitamin D in Advanced Non-Small Cell Lung Cancer Patients Treated with Nivolumab**

**Jessica Cusato <sup>1</sup> , Carlo Genova <sup>2</sup> , Cristina Tomasello <sup>3</sup> , Paolo Carrega 4,5, Selene Ottonello 6,7 , Gabriella Pietra 6,8, Maria Cristina Mingari 6,7,8, Irene Cossu <sup>9</sup> , Erika Rijavec <sup>10</sup>, Anna Leggieri <sup>3</sup> , Giovanni Di Perri <sup>1</sup> , Maria Giovanna Dal Bello <sup>10</sup>, Simona Coco <sup>10</sup>, Simona Boccardo <sup>10</sup> , Guido Ferlazzo 4,5,11, Francesco Grossi 2,\* ,† and Antonio D'Avolio 1,12,†**


Received: 11 December 2018; Accepted: 16 January 2019; Published: 21 January 2019

**Abstract:** Nivolumab is one of the most commonly used monoclonal antibodies for advanced non-small cell lung cancer treatment, to the extent that the presence of its anti-antibody is considered a negative prognostic factor. Vitamin D (VD) modulates expression of the genes involved in drug metabolism and elimination. Immune system regulation and immunodeficiency is frequent in non-small cell lung cancer patients. To date, no data have been reported about the relationship between nivolumab and VD. The aim of this study was to quantify plasma 25-hydroxyVD (25-VD) and 1,25-VD, nivolumab, and its anti-antibody before starting treatment (baseline) and at 15, 45 and 60 days of therapy. VD-pathway-associated gene single nucleotide polymorphisms (SNPs) were also evaluated. Molecules were quantified through enzyme-linked immunosorbent assay, and SNPs through real-time PCR. Forty-five patients were enrolled. Median nivolumab concentrations were 12.5 µg/mL, 22.3 µg/mL and 27.1 µg/mL at 15, 45 and 60 days respectively. No anti-nivolumab antibodies were found. Correlations were observed between nivolumab concentrations and 25-VD levels. Nivolumab concentrations were affected by VD-pathway-related gene SNPs. *VDBP* AC/CC genotype and baseline 25-VD < 10 ng/mL predicted a nivolumab concentration cut-off value of <18.7 µg/mL at 15 days, which was associated with tumor progression. This is the first study showing VD marker predictors of nivolumab concentrations in a real-life context of non-small cell lung cancer treatment.

**Keywords:** monoclonal antibody; NSCLC; immunotherapy; ELISA; pharmacokinetics; pharmacogenetics

#### **1. Introduction**

Immunotherapy represents the most revolutionary treatment for solid cancers nowadays. To date, several types of immunotherapy are available, including monoclonal antibodies, non-specific immunotherapies, oncolytic virus therapy, T-cell therapy and cancer vaccines. The evolution of immune checkpoint inhibitors as anticancer treatment options represents one of the most successful approaches in cancer drug research in the past few years [1]. Checkpoint inhibitor antibodies, such as anti-programmed cell death protein 1 (PD-1) and its ligand (PD-L1), are new drugs acting as tumor suppressing factors since they are able to modulate the interaction between the immune cell and the tumor cell [2]. These therapies proved to be a safe and effective option in advanced non-small cell lung cancer (NSCLC) and can be recommended selectively [3].

Nivolumab, a monoclonal antibody, binds to the immunomodulating PD-1, blocking ligand interaction and downstream signaling pathways. The result is a positive regulation of T-cell function resulting in an antitumor effect [4]. In 2015, this drug was approved by the FDA for the treatment of patients with advanced squamous and non-squamous NSCLC with progression, or after platinum-based chemotherapy (second-line therapy) [5]. In a randomized trial, 272 patients treated with nivolumab had an overall survival of 3.2 months longer than those on docetaxel [2].

In a conference abstract, authors measured nivolumab plasma concentrations in patients and suggested that partial responders had higher nivolumab mean trough concentrations (27.4 µg/mL) compared to subjects with tumor progression (18.7 µg/mL) [6].

PD-1 inhibitors typically cause fewer and less severe treatment-related adverse events (AEs) compared with conventional chemotherapy compounds, although immunorelated AEs can occur requiring monitoring and specialized management to prevent serious complications [7]. Moreover, immunogenicity in terms of the presence of nivolumab's anti-antibodies is considered a negative prognostic factor [8]. Immunogenicity and immune checkpoints in general are regulated by different factors such as vitamin D (VD) [9]. Reported studies show that VD controls different pathways related to innate and adaptive immunity regulating the expression of many genes involved in drug metabolism/elimination through its receptor (VDR). Moreover, in another study, single nucleotide polymorphisms (SNPs) in genes involved in the VD pathway could affect VD kinetics and, consequently, its action. Polymorphisms present near genes involved in cholesterol production, hydroxylation, and VD transport are able to predict who could have risk of VD insufficiency, as suggested by Wang et al. [10]. Genetic variations near DHCR7 (4p12 (overall *p* = 1.9 × 10(−109) for rs2282679, in GC); 11q12 (*p* = 2.1 × 10(−27) for rs12785878), near CYP2R1 (11p15 (*p* = 3.3 × 10(−20) for rs10741657) and near CYP24A1 (20q13)) have genome-wide significance in that population. Furthermore, participants with a score obtained combining the three variants in the highest quartile are at increased risk of 25-VD levels lower than 75 nmol/L or than 50 nmol/L, compared with those in the lowest quartile.

Since VD deficiency is frequent in lung cancer patients [11] and no data on nivolumab and its relationship with VD are currently available, the aim of this study was to quantify 25-hydroxyVD (25-VD), 1,25-hydroxyVD (1,25-VD), nivolumab, and its anti-antibody levels in patients' plasma at different timings, also considering their influence in predicting the cut-off value (18.7 µg/mL) associated with tumor progression.

#### **2. Results**

#### *2.1. Patient Characteristics*

Baseline (BL) characteristics for the 45 included patients are reported in Table 1. Thirty-one (69) were male, the median age was 73 years and the median body mass index (BMI) was 23.4 Kg/m<sup>2</sup> .


**Table 1.** Baseline characteristics of study population.

#### *2.2. Nivolumab and Vitamin D Concentrations*

Median nivolumab concentrations were 12.5 µg/mL (9.5–17.1 µg/mL), 22.3 µg/mL (IQR:18.30–34.88 µg/mL) and 27.1 µg/mL (IQR:17.4–39.4 µg/mL), respectively, at 15, 45, and 60 days (Figure 1). No anti-nivolumab antibodies were detected.

**Figure 1.** Nivolumab plasma concentrations at 15, 45 and 60 days.

The 25-VD concentration was 12.8 ng/mL (10.1–16.6 ng/mL), 13.6 ng/mL (10.9–16.1 ng/mL), 11.8 ng/mL (10.1–18.9 ng/mL), and 12.9 ng/mL (10.8–17.0 ng/mL) at BL, 15, 45, and 60 days, respectively.

The 1,25-VD value was 33.7 pg/mL (23.4–40.6 ng/mL), 34.7 ng/mL (22.3–45.4 ng/mL), 28.5 ng/mL (20.7–41.5 ng/mL), and 35.7 ng/mL (IQR:19.2–49.0 ng/mL), respectively, at BL, 15, 45, and 60 days.

Correlations (see Figure 2) were observed between nivolumab concentrations at 15 days and BL 25-VD levels (*p* = 0.024, Pearson's coefficient (PC) 0.451) and at 15 days (*p* = 0.017, PC = 0.542). Nivolumab exposure at 60 days was correlated with 25-VD at BL (*p* = 0.001, PC = 0.730), at 15 (*p* < 0.001, PC = 0.858), 45 (*p* = 0.001, PC = 0.779), and 60 days (*p* < 0.001, PC = 0.900). Furthermore, in a sub-group, patients were stratified according to 25-VD deficiency. BL 25-VD levels < 10 ng/mL were associated with lower nivolumab concentrations at 15 days (*p* = 0.103, a trend without statistical significance), 45 days (*p* = 0.018), and 60 days (*p* = 0.021). Fifteen days of 25-VD < 10 ng/mL levels were associated with lower nivolumab concentrations at 15 days (*p* = 0.019), 45 days (*p* = 0.019), and 60 days (*p* = 0.028). Finally, 60 days of 25-VD < 10 ng/mL was associated with lower nivolumab levels at 60 days (*p* = 0.030). No correlation was observed for 1,25-VD or toxicities and nivolumab exposure.

**Figure 2.** *Cont.*

**Figure 2.** Nivolumab and 25-hydroxyvitamin D correlations at different timings.

#### *2.3. Pharmacogenetics*

Variant genotype frequencies (%) were calculated and are reported in Table 2.



No genetic variants showed to affect VD concentrations. Nivolumab plasma concentrations at 15 days (Figure 3) were associated with *VDR* TaqI CC (*p* = 0.042), ApaI CA/AA (*p* = 0.030) and *CYP27B1*-1260 TT (*p* = 0.014). Nivolumab exposure at 45 days (Figure 4) were influenced by *VDR* Cdx2 AG/GG (*p* = 0.019), *VDBP* rs7041 AC/CC (*p* = 0.035), and *CYP27B1*-1260 TT (*p* = 0.028); nivolumab exposure at 60 days (Figure 5) was affected by *VDR* Cdx2 AG/GG (*p* = 0.022) and TaqI TC/CC (*p* = 0.021). VDR: vitamin D receptor.

**Figure 3.** Influence of gene variants on nivolumab plasma concentrations at 15 days.

**Figure 4.** Influence of gene variants on nivolumab plasma concentrations at 45 days.

**Figure 5.** Influence of gene variants on nivolumab plasma concentrations at 60 days.

#### *2.4. Regression Analysis*

A logistical regression analysis was performed to evaluate whether factors (demographic, clinical, pharmacological or genetic) were able to predict nivolumab concentrations <18.7 µg/mL at 15 days (see Table 3). According to a Bonferroni test, *p* < 0.003 was considered to be the adjusted *p*-value, but no factors reached this value in the univariate analysis. In the multivariate model, *VDBP* (GC) AC/CC genotype and BL 25-VD were predictors of this cut-off value, associated with tumor progression (Figure 6).


**Table 3.** Logistic regression analyses: Factors able to predict nivolumab concentrations <18.7 µg/mL at 15 days of therapy. Bold represents statistically significant values. NC: not comparable, all the factors belong to a single group. Thus, statistics could show *p*-values and *odd-ratio* (OR).

**Figure 6.** *VDBP* rs7041 SNP and pre-25 hydroxyvitamin D levels predictors of the nivolumab cut-off value of 18.7 µg/mL at 15 days, associated with tumor progression.

#### **3. Discussion**

Nivolumab represents an active treatment strategy with the potential of long-term disease control [12]. Unfortunately, biomarkers of reliable efficacy are lacking, thus nivolumab has not been considered to be cost-effective in several national health systems [13,14].

However, a meta-analysis [3] on immune checkpoint inhibitors and chemotherapy in the treatment of advanced NSCLC showed significant advantages in terms of overall survival, progression-free survival, and overall response rate, compared with conventional chemotherapy in patients with advanced disease.

VD is able to regulate the immune system. Its synthesis begins by the action of ultraviolet light in the context of skin tissue. Cholecalciferol is hydroxylated to calcifediol (25-VD) in the liver through cytochrome P-450 (CYP, 27A1, 2R1). In the kidney, calcitriol (1,25-VD, the active form) is synthesized through CYP27B1 and transported in the bloodstream through vitamin D binding protein (VDBP). The inactivation of 25-VD to calcitroic acid (24,25-VD) is carried on by CYP24A1. VD deficiency is frequently observed in cancer patients. Bochen et al. suggested that VD serum levels were significantly lower in head and neck cancer patients compared to controls, particularly in patients with lymphatic metastasis [15]. Different studies show that a lower 25-VD serum level is associated with several negative outcomes in lung cancer. Feng et al. analyzed seventeen studies in a meta-analysis and found a statistically significant relationship between 25-VD, lung cancer risk, and mortality, but a relationship with overall lung cancer survival was not observed [16]. In addition, they suggested differences between males and females and in Caucasian and Asian populations in terms of cancer risk.

In the current study, 25-VD influenced nivolumab concentrations, but not 1,25-VD. Here, we only evaluate nivolumab and VD concentrations and not the effect on the immune cells. VD deficiency could have a relapse due to the immune system, which is directly related to this treatment. In fact, in another study, a relationship between immune cells and 25-VD and not with 1,25-VD was found, as shown for regulatory T cell function in multiple sclerosis patients [17]. Information about the influence of VD on the immune system is lacking in this study. This limitation will be the aim of further studies by our group.

Furthermore, 1,25-VD is present with a concentration 1000 times lower than 25-VD in the blood. Such low 1,25-VD concentrations could be more difficult to measure compared to 25-VD levels. Finally, the absence of statistical significance could be due to the small sample size.

In the current study, the nivolumab plasma levels in a real-life context of NSCLC are described at different timings and, in addition, the role of 25-VD concentrations and *VDBP* rs7041 A > C SNP in predicting concentrations lower than 18.7 µg/mL (the cut-off value associated with tumor progression as shown by Stijn et al. [6]) is suggested.

Various *VDBP* genetic variants are known. The two most common polymorphisms, 1296 A > C (rs7041, Glu432Asp) and 1307 C > A (rs4588, Thr436Lys), are localized in exon 11 and they are in complete linkage disequilibrium [18]. Circulating VDBP seems not to be influenced by rs7041 SNP, however, considering the 1296/1307 diplotype, there is a slight transport increase in AC/CA, compared to AA/CA. It is probable that lysine to threonine substitution at position 436 eliminates an O-glycosylation site from the molecule and the loss of glycosylation influences the half-life of VDBP. Moreover, glutamine to asparagine changes in the 432 position affect the extent of O-glycosylation at the 436. It is not known how changes in the VDBP molecule modify its serum concentration, but the described substitutions could result in altered rates of transcription, changes in mRNA stability, or in a self-clearance of the protein [19]. In a recent study of Caucasian women, the AA genotype was related to higher breast cancer risk, compared to healthy controls [20].

Controversial studies are present in the literature concerning the influence of VDBP rs7041 on VD levels. Lafi et al. show that genotypes containing the variant allele of rs7041 (TT, TG) are associated with lower 25-VD concentrations than the GG genotype, whereas Daffara et al. did not find an association in coronary heart disease patients and suggest that 25-VD levels, but not VDBP genetic status, independently predicted the presence of coronary lesions at angiography [21,22]. Also, in the current study, an association between the *VDBP* genetic variant and VD levels has been evidenced, although a borderline influence (*p* = 0.049) is present with the nivolumab cut-off value. However, the best predicting factor remains 25-VD < 10 ng/mL, as showed in the regression. It is important to understand the nature of the relationship between these variables: is the VD associated with poorer outcomes, or it could be an underlying condition? In our opinion, VD deficiency could be able to affect the outcome, since it is involved in the regulation of the immune system. Furthermore, in deficient individuals before starting therapy, the situation could be more difficult to manage and complications could be more severe (for example, concerning cachexia).

Schmid et al. showed that immunotherapy efficacy was dependent on the metastatic location [23]. For these reasons, it is very important to understand which biomarkers could predict patients with a higher probability of tumor progression.

Our study would recommend to clinicians to evaluate 25-VD levels and the *VDBP* rs7041 genotype, before starting therapy, and to quantify nivolumab concentrations at 15 days, to eventually consider a drug dosage modification or VD supplementation, reducing the risk of tumor progression. It is important to highlight that these analyses are preliminary and have several limitations: They are conducted on few individuals (only 45 patients), only one cohort is analyzed, and *VDBP* SNP has a borderline influence (*p* = 0.049).

#### **4. Materials and Methods**

Patients were treated with nivolumab, affected by advanced NSCLC, treated within the Italian Nivolumab Expanded Access Program (NCT02475382), and enrolled in a mono-institutional translational research study at the Lung Cancer Unit of the Ospedale San Martino (Genova, Italy). This study was approved by the Local Ethics Committee (registry number: P.R. 191REG2015). Patients were eligible if they met the following criteria: (i) cytologically or histologically confirmed advanced/metastatic NSCLC, (ii) progression after at least one line of platinum-based chemotherapy, (iii) Eastern Cooperative Oncology Group Performance Status (ECOG-PS) = 0–2, (iv) no previous treatment with immune checkpoint inhibitors, (v) any brain metastasis had to be treated and clinically stable for at least 14 days before starting nivolumab, (vi) no treatment with corticosteroids at a dose higher than 10 mg/day of prednisone or equivalent. Eligible patients received nivolumab at 3 mg/kg every 14 days, with assessment by computed tomography scan (CT-scan) every 8 weeks. Nivolumab was administered until the onset of unacceptable toxicities, patient refusal, death, or up to 96 weeks from the start of treatment. Treatment beyond tumor progression was allowed based on the investigators' judgment, as long as clinical benefit was perceived.

Values of 25-VD and 1,25-VD were evaluated at BL and at 15, 45, and 60 days after starting therapy, with enzyme-linked immunosorbent assay technique (DRG DIAGNOSTIC, Marburg, Germany) and with LIAISON® XL (DiaSorin, Saluggia, Italy), respectively. Nivolumab and its anti-antibody were quantified with validated ELISA kits (Matrix Biotek, Ankara, Turkey).

Whole blood was drawn in EDTA tubes, genomic DNA was isolated from blood samples (MagnaPure Compact, Roche, Monza, Italy), and genotypes were assessed through a real-time polymerase chain reaction allelic discrimination system (LightCycler 480, Roche, Monza, Italy). The investigated gene SNPs were: *CYP27B1* (encoding cytochrome 27B1 enzyme responsible for VD active metabolite 1,25-VD production) rs4646536 (+2838) C > T and rs10877012 (−1260) G > T, *VDR* (encoding VD receptor) rs7975232 (ApaI) C > A, rs731236 (TaqI) T > C, rs10735810 (FokI) T > C, rs11568820 (Cdx2) A > G and rs1544410 (BsmI) G > A, *CYP24A1* (encoding cytochrome 27B1 enzyme responsible for VD inactive metabolite 24,25-dyhydroxyvitamin D (24,25-VD) production) rs2248359 (3999) T > C, rs927650 (22776) C > T and rs2585428 (8620) A > G and finally *GC* (encoding VD transporter, VDBP) rs7041 A > C.

The analysis of PD-L1 was performed in 29 out of 45 patients with available tumor tissue at diagnosis using Immunohistochemistry. In particular, the PD-L1 expression was assessed manually using the rabbit monoclonal anti-human PD-L1 antibody clone 28-8 (Pharm DX DAKO, CA, USA), according to the FDA approved auto-stainer link 48 protocol. The tumor samples were defined as positive when at least 1% of tumor cells showed a strong staining according to their membrane location. All variables were tested for normality through the Shapiro–Wilk test. Normal variables were described as average and standard deviation, non-normal variables as median values and interquartile range (IQR), and categorical variables as numbers and percentages. Allele frequencies were tested for Hardy–Weinberg equilibrium. Kruskal–Wallis and Mann–Whitney tests were adopted for differences in continuous variables between genetic groups, considering statistical significance with a two-sided *p*-value < 0.05. Stepwise multivariate logistic regression analysis was performed including variables with a *p*-value below 0.2 at univariate analysis to evaluate whether factors were able to predict nivolumab levels <18.7 µg/mL at 15 days. A Bonferroni correction was performed, since an adjustment made to *p*-values is needed when several dependent or independent statistical tests are being performed simultaneously on a single data set [24].

Tests were performed using IBM SPSS Statistics 25.0 for Windows (Chicago, IL, USA).

#### **5. Conclusions**

In conclusion, this is the first study showing an association between VD-related biomarkers and nivolumab plasma concentrations.

In the current study, for the first time, VD deficiency seems to result in altered nivolumab clearance, as shown by different associations. It is interesting to highlight that, according to these analyses, the reduction in VD concentration was not through antibodies.

Future studies will aim to analyze the effect of VD deficiency on the immune system, for example, evaluating the immunologic profile according to VD-related biomarkers or PD-1 or PD-L1 levels and their genetic variants.

These are preliminary and limited analyses, and further studies in larger and different cohorts are needed to clarify these aspects, and to improve the knowledge in the field of the monoclonal antibody treatment used in NSCLC.

**Author Contributions:** J.C. conceived and directed the project, performed vitamin D and nivolumab quantification, the statistical analyses and wrote the article; C.G. enrolled patients, follwed up during treatment, recovered all clinical data and contributed to the writing of the article; C.T. performed vitamin D and nivolumab quantification and wrote the article; P.C. organized the sample collection, processed the serum at the diffent timepoints, contributed to data obtainment and the writing of the article; S.O. processed sample at the different timepoints, G.P. processed the serum at the diffent timepoints; M.C.M. contributed to the data collection; I.C. processed the serum at the diffent timepoints; E.R. contributed to the data collection; A.L. evaluated the pharmacoeconomical impact of this study; G.D.P. contributed to the acquisition of the fundings; M.G.D.B. and S.C. coordinateed the sample collection and recorded the clinical information in database for analyses; S.B. performed the immunohistochemistry and data interpretation for PD-L1 expression; G.F. contributed to the organization of the study; F.G. supervised the project, contributed to the design of the research, to the analysis of the results and to the writing of the manuscript; A.D. supervised the project, contributed to the acquisition of the fundings, to the design of the research, to the analysis of the results and to the writing of the manuscript. All authors discussed the results, rviewed and approved the manuscript.

**Funding:** C.G. received honoraria from Astra Zeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Merck Sharp and Dohme, Roche; E.R. received honoraria from Astra Zeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Roche; F.G. received honoraria from AMGEN, Astra Zeneca, Bristol-Myers Squibb, Boehringer Ingelheim, Celgene, Merck Sharp and Dohme, Pfizer, Pierre Fabre, Roche.The PD-L1 analysis was supported by a grant from Compagnia San Paolo (SC: 2017-0529).

**Acknowledgments:** We thank CoQua Lab (www.coqualab.it) for its methodological support and assistance in the preparation and execution of the study and analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Tumor Neovascularization and Developments in Therapeutics**

**Yuki Katayama, Junji Uchino \* , Yusuke Chihara, Nobuyo Tamiya, Yoshiko Kaneko , Tadaaki Yamada and Koichi Takayama**

Department of Pulmonary Medicine, Kyoto Prefectural University of Medicine, 465 Kajiicho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan; ktym2487@koto.kpu-m.ac.jp (Y.K.); c1981311@koto.kpu-m.ac.jp (Y.C.); koma@koto.kpu-m.ac.jp (N.T.); kaneko-y@koto.kpu-m.ac.jp (Y.K.); tayamada@koto.kpu-m.ac.jp (T.Y.); takayama@koto.kpu-m.ac.jp (K.T.) **\*** Correspondence: uchino@koto.kpu-m.ac.jp; Tel.: +81-75-251-5513

Received: 28 December 2018; Accepted: 4 March 2019; Published: 6 March 2019

**Abstract:** Tumors undergo fast neovascularization to support the rapid proliferation of cancer cells. Vasculature in tumors, unlike that in wound healing, is immature and affects the tumor microenvironment, resulting in hypoxia, acidosis, glucose starvation, immune cell infiltration, and decreased activity, all of which promote cancer progression, metastasis, and drug resistance. This innate defect of tumor vasculature can however represent a useful therapeutic target. Angiogenesis inhibitors targeting tumor vascular endothelial cells important for angiogenesis have attracted attention as cancer therapy agents that utilize features of the tumor microenvironment. While angiogenesis inhibitors have the advantage of targeting neovascularization factors common to all cancer types, some limitations to their deployment have emerged. Further understanding of the mechanism of tumor angiogenesis may contribute to the development of new antiangiogenic therapeutic approaches to control tumor invasion and metastasis. This review discusses the mechanism of tumor angiogenesis as well as angiogenesis inhibition therapy with antiangiogenic agents.

**Keywords:** cancer therapy; neovascularization; angiogenesis; tumor microenvironment

#### **1. Introduction**

Vasculogenesis refers to the process by which vascular endothelial cells differentiate from endothelial precursor cells to form the lumen. Neovascularization refers to the process, whereby new blood vessels are formed from existing ones following endothelial cell proliferation and migration [1]. This process is essential during physiological angiogenesis, such as systemic blood supply in the fetal stage, luteinization related to postpartum menstrual cycle, and wound healing [2]. During tumor proliferation, oxygen and nutrients required for solid tumor growth are supplied from neighboring blood capillaries. However, because the diffusion distance of oxygen is 100–200 µm, for tumors to grow to ≥1–2 mm, generation of new blood vessels towards the tumor (i.e., neovascularization) is required [3,4]. Tumors located >100–200 µm from capillaries often encounter hypoxic conditions, which promote the expression of hypoxia-inducible factor-1 (HIF-1). HIF-1 induces the expression of angiogenic proteins, such as vascular endothelial growth factor (VEGF), epidermal growth factor, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and platelet-derived growth factor (PDGF), which then stimulate hypervascularization [5,6]. The sustained expression of these angiogenic factors results in abnormally structured angiogenic tumor vessels. Tortuous and dilated tumor vessels show increased vascular permeability and high interstitial pressure, further reducing blood perfusion and increasing hypoxic conditions in the tumor microenvironment [7–9]. Administration of

angiogenesis inhibitors leads to tumor vascular normalization, a reduction in vascular permeability and interstitial fluid pressure, and an improvement in tumor perfusion. A normalized tumor vascular system with reduced hypoxic conditions not only augments the effects of radiotherapy and chemotherapy but also enhances antitumor immunity [10–12]. The findings can contribute to a new approach (i.e., the combination of angiogenesis inhibitors and immunotherapy) to further improve the overall survival of cancer patients. This review discusses the molecular mechanisms of tumor angiogenesis and outlines options for cancer therapy with antiangiogenic agents including combined immunotherapy.

#### **2. Molecules Involved in Neovascularization**

Neovascularization is regulated by a balance between angiogenesis-inducing factors and angiogenesis-inhibiting factors such as those outlined in Table 1. Here, we describe the molecules that induce angiogenesis and their mechanisms. Among angiogenesis-inducing factors, VEGF plays an important role in the initiation of angiogenesis. The VEGF family consists of five members, namely VEGFA, VEGFB, VEGFC, VEGFD, and placental growth factor (P1GF). VEGF signals are transmitted through three VEGF receptor tyrosine kinases: VEGFR1, VEGFR2, and VEGFR3 [8,13]. The VEGF family of proteins is the most critical factor for the induction of neovascularization. VEGF induces proliferation of endothelial cells, promotes cell migration, and decreases the rate of apoptosis. It also increases vascular permeability and promotes migration and circulation of other cells [13,14]. VEGFA and its receptor, VEGFR2, have major angiogenic effects [15]. Upon binding to the VEGF receptor on the vascular endothelial cell membrane, VEGF induces dimerization and autophosphorylation of the receptor and initiates a signaling cascade that activates a variety of downstream pathways. Phosphorylation of phospholipase C (PLC) γ activates the RAS/mitogen-activated protein kinase (MAPK) cascade via protein kinase C (PKC) activation and regulates gene expression and cell proliferation [16–18]. In addition, activation of the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway produces NO via AKT, suppresses apoptosis, and activates endothelial cell NO synthase, thereby enhancing vascular permeability [19–22]. VEGFR1 has a weak kinase activity and limits VEGFR2-induced angiogenic effects by regulating the amount of VEGFA that can be bound by VEGFR2 [23]. The following has been reported: (i) VEGFR3 and its ligand, VEGFC, are responsible for lymphangiogenesis; (ii) VEGFC and VEGFD contribute to tumor angiogenesis by binding to VEGFR2 and VEGFR3; (iii) VEGFR3 is expressed in the tip cells of tumor vessels [15,24].


**Table 1.** Endogenous regulators of angiogenesis.

Angiopoietins play a critical role in the maturation of blood vessels. Human angiopoietins consist of three ligands, Ang-1, Ang-2, and Ang-4. Ang-1 and Ang-2 are of critical importance in angiogenesis and are outlined hereafter. The Tie family of receptors includes receptor tyrosine kinases specifically expressed in the vascular endothelium. They include Tie1 and Tie2. Tie2 is activated by Ang-1, which is secreted by platelets and peri-endothelial cells; whereas Tie1 is an orphan receptor homolog of Tie2, whose expression enhances Tie2 activation [25]. The Ang-1/Tie-2 signaling pathway is specific for endothelial cells. Ang-1 binds to the Tie-2 tyrosine kinase receptor of endothelial cells, whose downstream phosphorylation activity stimulates cell survival by activating the PI3K-AKT pathway [26–28]. Furthermore, it contributes to the maturation of blood vessels by inhibiting the proinflammatory pathway initiated by nuclear factor-kappa B (NF-κβ) [26–28]. In contrast, in the absence of cell-cell adhesion, extracellular matrix-anchored Tie2 regulates angiogenesis via extracellular signal-regulated kinase (ERK) 1/2 signaling [29]. Ang-2 antagonizes Ang-1 activity and, in the presence of low levels of VEGF, leads to detachment of pericytes and regression of blood vessels. However, in the presence of high levels of VEGF, Ang-2 elicits an inflammatory response and destabilizes existing vessels. This, in turn, promotes angiogenesis and lymphangiogenesis by weakening the interaction between endothelial cells and pericytes and increasing endothelial cell migration [1,30,31].

#### **3. Characteristics of Angiogenic Tumor Vessels**

Angiogenesis-promoting factors such as VEGF induced by the tumor microenvironment (e.g., hypoxia), stimulate sustained and abnormal neovascularization [32,33]. The vessels formed during neovascularization are unlike those formed during wound healing and exhibit unusual morphological characteristics. In normal vessels, the distribution of arteries, capillaries, and veins is stable, and the vessels have an ordered hierarchical structure. In comparison, angiogenic tumor vessels are dilated and tortuous. Furthermore, vascular density and blood vessel diameter are not uniform [34,35]. A simple squamous epithelium, known as vascular endothelial cells, covers the lumen of capillaries, which is lined with pericytes and covered by the basement membrane. Angiogenesis promoting factors induce weakening and migration of vascular endothelial cell junctions and change the vascular wall structure [36,37]. Pericytes and vascular endothelial cell junctions between pericytes and vascular smooth muscle cells are also weakened, and the number of pericytes is reduced [38,39]. The basement membranes are multilayered and collagen IV thickness is uneven. Weakened cell junctions between endothelial cells and pericytes result in their infiltration into the tumor stroma [38,39]. The morphological abnormalities observed in tumor blood vessels raise the question of whether there are phenotypic differences at the molecular and functional levels between tumor endothelial cells (TECs) that line tumor blood vessels and normal endothelial cells. TECs express higher levels of proangiogenic genes such as VEGFR, VEGF, and EGFR. The Hu antigen, a neuronal protein identified in the serum of patients with small cell lung cancer and paraneoplastic encephalomyelitis/sensory neuronopathy, promotes TECs survival by stabilizing VEGF mRNA. TECs also up-regulates integrin αVβ3 and cause cytogenetic abnormalities [40]. Moreover, in comparison with normal endothelial cells, TECs have a high proliferative capacity, migratory ability, and angiogenic potential [41]. Additionally, cells showing stem cell/precursor cell-like properties have been reported in the TECs population, together with those originating from bone marrow-derived vascular endothelial progenitor cells and tissues derived from tissue stem cells [42]. Furthermore, a population expressing stem cell markers such as aldehyde dehydrogenase and having high angiogenic potential has also been reported [43]. ATP-binding cassette sub-family B member 1 (ABCB1) is the most well-known drug efflux transporter and TECs strongly expressing ABCB1 are resistant to drugs [44]. Importantly, cancer microenvironment factors such as hypoxia, are also thought to be involved in tumor vascular endothelial cell abnormalities, together with humoral factors derived from cancer cells and exosomes [45,46]. Tumor vascular endothelial markers are expressed in cancer cells when cultured under hypoxia or in a low-serum medium. Furthermore, Kubota et al. reported that an ataxia telangiectasia mutated kinase was strongly activated in immature vessels in response to the accumulation of reactive oxygen species, where it provided

a defensive function [47]. More recently, Maishi et al. demonstrated that biglycan secreted by TECs induced intravascular invasion and metastasis of cancer cells and reported a new mechanism of cancer metastasis induction from tumor vessels [48]. As can be seen, various factors typical of the cancer microenvironment exert complex and diverse properties in tumor vascular endothelial cells.

#### **4. Regulatory Mechanisms of Neovascularization**

### *4.1. HIF-1α*

Hypoxic conditions in the tumor microenvironment up-regulate angiogenesis inducing factors such as VEGF, PDGF, P1GF, and HGF. However, reactions activated by such a hypoxic environment are thought to be elicited primarily by HIF-1α [49,50]. HIF is a transcription factor heterodimer consisting of subunits HIF-1α and HIF-1β [51,52]. When oxygen tension is normal, HIF-1α is quickly degraded [53]. Under normal oxygen concentration, HIF-1α is modified by prolyl 4 hydroxylase (PHD), which acts as a direct oxygen sensor by catalyzing the binding of molecular oxygen to a specific proline on HIF-1α [52]. The Von Hippel-Lindau cancer suppression protein binds to hydroxylated HIF-1α to activate the protein complex and targets HIF-1α for proteasome-dependent degradation following its ubiquitination [54]. Under normal oxygen conditions, asparagine residues near the C-terminus are hydroxylated by factor inhibiting HIF-1 (FIH-1), which also requires oxygen for its activity. FIH-1 reduces HIF-1α transcriptional activity by preventing the binding of p300 and cAMP response element binding protein (CREB) to HIF-α [55,56]. As PHD is inactive and HIF-1α is not hydroxylated in a low-oxygen environment, the Von Hippel-Lindau factor cannot bind and direct HIF-1α for proteasome-mediated proteolysis. Instead, HIF-1α can bind to p300 and CREB. The HIF-1α-conjugated protein is also believed to be transferred to the nucleus, heterodimerized by HIF-1β, and immediately involved in initiating transcription of target genes. With a binding site corresponding to 5′ -RCGTG-3′ , the HIF heterodimer transcription factor activates target genes via a hypoxia response sequence (HRE) [57]. HIF-1α binds to the HRE of VEGFA, PDGF, and transforming growth factor-alpha, inducing their expression (Figure 1) [58].

In addition to angiogenesis, HIF-1α activates glucose metabolism, thereby leading to acidosis in the tumor microenvironment. HIF-1α enhances the expression of glucose transporter 1, 3 and increases the uptake of glucose into cells [59]. Additionally, it cleaves glycolytic enzymes (phosphofructokinase L, hexokinase, aldolase A, and lactate dehydrogenase A) by activating ATP production and promoting glycolysis [60,61]. HIF-1α activates pyruvate dehydrogenase kinase 1, which then inactivates pyruvate dehydrogenase, resulting in suppression of the TCA cycle [62]. Thus, overexpression of HIF-1α under hypoxic conditions accelerates lactic acid production by promoting glycolysis and suppressing the TCA cycle, leading to an acidic tumor microenvironment. The latter contributes to tumor survival by conferring apoptosis resistance [63], increasing invasion and metastatic potential [64], and providing immune tolerance through T cell suppression [65].

**Figure 1.** Hypoxia inducible factor (HIF) and vascular endothelial growth factor (VEGF) link the angiogenesis signaling pathways. Low oxygen tension (hypoxia) results in constitutive activation of the HIF pathway and VEGF. The tumor hypoxic environment leads to an immunosuppressive tumor microenvironment by inducing regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and M2 tumor-associated macrophages (TAMs). Antiangiogenic therapy results in blood vessel regression by suppression of neovascularization, leading to tumor starvation and tumors falling into dormant states. CAFs, cancer-associated fibroblasts; iDCs, immature dendritic cells; mDCs, mature dendritic cells; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion molecule 1.

#### *4.2. Endoplasmic Reticulum Stress Signals*

The endoplasmic reticulum is a protein folding and maturation site within the cell, where membrane proteins are glycosylated and secreted. Failure to mature results in the accumulation of proteins with an abnormal higher-order structure. Accumulation of such abnormal proteins causes endoplasmic reticulum stress, and the cellular responses elicited to deal with it are collectively referred to as the endoplasmic reticulum stress response. Tumor microenvironment characteristics such as hypoxia, acidosis, and glucose deprivation contribute to the activation of the endoplasmic reticulum stress pathway and promote cancer cell survival. Up-regulation of the endoplasmic reticulum molecular chaperone, BiP/GRP78 has been observed in multiple cancer cells, indicating that it is involved in their proliferation and metastasis [66]. VEGF expression is induced in the protein kinase R like endoplasmic reticulum kinase (PERK)-activating transcription factor 4 (ATF4) pathway in the endoplasmic reticulum stress environment [67]. The inositol-requiring kinase enzyme 1 alpha (IRE1α) signal also promotes cell growth in certain cancer cell types due to up-regulation of cyclin A1 via X-box binding protein 1 (XBP-1) downstream of IRE1α [68]. Expression of XBP-1 is elevated in breast cancer cells and hepatocellular carcinoma. Therefore, XBP-1 expression is believed to contribute to the survival of cancer cells by inducing BiP/GRP78 expression [69]. As in the case of PERK and IRE1α signals, ATF6 is thought to be involved in neovascularization by controlling the expression of VEGF [70]. In addition to cancer cells, tumor tissues and their microenvironments include fibroblasts, mesenchymal stem cells, and immune cells including macrophages and T cells. Cells that build these tumor microenvironments can induce angiogenesis by producing multiple growth factors, cytokines, and chemokines. Fibroblasts in tumor tissue are the major constituents of tumor stromal tissue and are said to play a vital role in cancer development. Known as cancer-associated fibroblasts (CAFs) they secrete stromal

cell-derived factor 1 (SDF1). CAF-derived SDF1 not only directly stimulates cancer cell proliferation via C-X-C chemokine receptor type 4 on tumor cells, but also recruits endothelial progenitor cells towards the tumor and induces angiogenesis [71]. In colorectal cancer, angiogenesis is promoted by CAF-induced secretion of interleukin 6 and a concomitant increase in VEGF production [72]. Macrophages involved in carcinogenesis and malignancy are called tumor-associated macrophages (TAMs). Most TAMs are composed of M2 macrophages, which affect tumor development through increased immunosuppression and angiogenesis. TAMs stimulate angiogenesis directly by facilitating the production of angiogenesis promoting factors such as VEGF, and indirectly by localizing matrix metallopeptidase 9 to the tumor microenvironment. There, metallopeptidase 9 induces angiogenesis by cleaving and releasing VEGF from the matrix [73,74]. In addition, vascular endothelial cells produce Ang-2c and TAMs express its receptor, Tie2, further stimulating angiogenesis in tumor tissues [75].

#### **5. Antiangiogenic Therapy**

Clinical treatment approaches targeting tumor angiogenesis include the anti-VEGF monoclonal antibody bevacizumab, anti-VEGFR2 monoclonal antibody ramucirumab, VEGFR ligand traps (e.g., aflibercept, VEGFR, PDGFR, c-KIT), and multi-target tyrosine kinase inhibitors (e.g., sunitinib and sorafenib) [76,77] (Table 2, Figure 2). Bevacizumab, a humanized monoclonal immunoglobulin G1 antibody, is the most widely studied antiangiogenic agent that prevents VEGFA from binding to receptors, thus hindering neovascularization and the activation of signal transduction cascades [78]. After bevacizumab combined with chemotherapy was first approved by the U.S. Food and Drug Administration (FDA) in 2004, the drug was also approved by the FDA for use in non-small cell lung cancer, metastatic colorectal cancer, renal cell carcinoma (RCC), ovarian cancer, glioblastoma multiforme, cervical cancer, fallopian tube cancer, and primary peritoneal cancer [79].


**Table 2.** Angiogenesis inhibitors approved by FDA.

Ramucirumab combined with chemotherapy has been shown to extend overall survival of gastric cancer, non-small cell lung cancer, and rectal cancer patients. In 2012, aflibercept (i.e., a peptide-antibody fusion targeting the VEGF ligand) combined with fluorouracil, irinotecan, and folinic acid was also approved by the FDA for use in colorectal cancer [80]. Sunitinib and sorafenib, which are multi-target tyrosine kinase inhibitors, have been approved as monotherapy agents based on improvement in overall survival and progression-free survival in phase III studies in metastatic-differentiated thyroid cancer, unresectable hepatocellular carcinoma, and advanced RCC [81–83].

**Figure 2.** Vascular endothelial growth factor (VEGF) binds to the VEGF receptor, a receptor tyrosine kinase, leading to receptor dimerization and subsequent auto phosphorylation of the receptor complex. The phosphorylated receptor then interacts with a variety of cytoplasmic signaling molecules, leading to signal transduction and eventually angiogenesis. Examples of clinical drugs (Table 2) that inhibit the pathway are shown. PI3K, phosphoinositide-3-kinase; AKT, protein kinase B; mTOR, mechanistic target of rapamycin; MEK, MAPK/ERK kinase; ERK, extracellular signal-regulated kinase.

#### **6. Resistance Mechanism of Angiogenesis Inhibitors**

Resistance to angiogenesis inhibitors develops through a variety of mechanisms such as activation of an alternate angiogenic pathway that promotes tumor angiogenesis. When VEGF and VEGFR are inhibited, other angiogenic factors such as P1GF, SDF1, Ang-1, FGF, HGF, and cytokines, are induced [84]. In preclinical models, FGF1, FGF2, Ang-1, Ephrin-A1, and Ephrin-A2 have been induced in pancreatic tumors treated with anti-VEGFR2 antibody [85]. HGF, bFGF, and P1GF levels were increased in patients with metastatic colorectal cancer before disease progression when treated with a combination of fluorouracil, irinotecan, and bevacizumab [86]. Cancers such as colorectal cancer, RCC, and neuroendocrine tumors are often highly dependent on the induction of angiogenesis by VEGF. On the opposite end, cancers that are less susceptible to anti-VEGF antibodies, such as breast cancer, pancreatic cancer, malignant melanoma or prostate cancer, use different angiogenesis mechanisms and angiogenic factors [87]. Long-term administration of angiogenesis inhibitors induces hypoxia in the tumor microenvironment by over-pruning blood vessels and up-regulates HIF-1α [88]. Angiogenesis promoting factors, such as P1GF, VEGF, Ang-1, and FGF, which are induced by HIF-1α, recruit bone marrow-derived dendritic cells (BMDCs) that mediate the growth of new blood vessels to support tumors. The presence of BMDCs in the tumor environment induces resistance to angiogenesis inhibition [89,90]. In addition to BMDCs, the hypoxic environment within the tumor promotes recruitment of regulatory T cells (Tregs), bone marrow-derived repression cells (MDSCs), and M2 TAMs.

Immune cell populations in tumors promote angiogenesis, tumor growth, epithelial-mesenchymal transition, metastasis, and immunosuppression of the tumor microenvironment [91,92]. Besides acquiring resistance to angiogenesis inhibition by inducing other cells, tumor cells have also been reported to escape the effect of angiogenesis inhibitors by adopting different neovascularization modalities, including vascular co-option and vasculogenic mimicry [93]. Vessel co-option refers to the process whereby cancer cells incorporate pre-existing vessels from surrounding tissue instead of inducing new vessel growth [94]. The main factors regulating vessel co-option are VEGF and angiopoietins. Moreover, several studies have reported an increase in vessel co-option after inhibition of angiogenesis [95]. Anti-VEGF antibody treatment in glioblastoma promotes an increase in vessel co-option, and similar phenomena have been reported in other solid tumors [96]. Vasculogenic mimicry refers to a situation, whereby tumor cells function like endothelial cells and form a blood vessel-like structure [97]. This phenomenon has been reported in malignant melanoma, sarcoma, glioma, breast cancer, and many other cancer types [98–100]. Preclinical studies have reported increased vasculogenic mimicry by angiogenesis inhibition therapy with bevacizumab, and the effectiveness of combining angiogenesis inhibitors with chemotherapy has been suggested.

#### **7. Neovascularization and Immunity**

Tumor angiogenesis and tumor immunity share a complex relationship. When exposed to hypoperfusion/vascular hyperpermeability by immature tumor neovasculature, the tumor microenvironments becomes hypoxic and VEGF is up-regulated. This induces a decrease in T cell activation by dendritic cells (DCs), a reduction in the number of intratumorally infiltrating lymphocytes, and an increase in immunosuppressive cells, all of which affect immune function [101]. Steady-state immature dendritic cells (iDCs) in vivo are superior in antigen uptake ability, but have weak T cell stimulating ability and induce immune tolerance through Treg activity. iDCs that phagocytose and process the antigen, migrate to regional lymph nodes where they convert to mature dendritic cells (mDCs) that present the antigen to T cells and activate them [102]. Although DC maturation is activated by the NF-κB pathway, the increase in VEGF due to the hypoxic environment of the tumor reduces the number of mDCs by inhibiting DC maturation through inhibition of the NF-κB pathway and suppresses immunity [103–105]. Furthermore, VEGF binds to VEGFR2, inhibits the T cell activation function of mDCs, up-regulates the expression of programmed cell death ligand 1 (PD-L1) (B7-H1/CD274), and suppresses the function of DCs [106]. The migration and adhesion of vascular adhesion molecules to vascular endothelial cells plays an important role in the activation of immunity by causing the accumulation of immune cells, such as macrophages, NK cells, granulocytes, B cells, and T cells [107]. VEGF promotes abnormal neovascularization and affects immune cell migration, which reduces the expression of cell adhesion molecules, such as intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin. The down-regulation of cell adhesion molecules inhibits tumor invasion by immune cells and reduces the immune response [108–110]. A tumor immune response is induced by tilting the quantitative and functional balance of tumor-attacking effector T cells and immunosuppressive cells to the former dominant state. The tumor hypoxic environment enhances the expression of SDF1-α and C-C motif chemokine 28, thereby inducing immunosuppressive cells such as Tregs, MDSCs, and M2 TAMs, and suppresses tumor immunity [91,92,111]. When VEGF binds to VEGFR on MDSCs, signal transducer and activator of transcription 3 signaling is activated and induces MDSC proliferation [112], VEGF also promotes an increase in Tregs in the tumor microenvironment [113,114]. Increasing the recruitment of T cells and promoting tumor invasion by angiogenesis inhibitors have shown the effect of tilting the tumor microenvironment towards immunity promotion. Bevacizumab and sorafenib induce DC maturation and improve T cell activation [115]. Inhibition of VEGF increases E-selectin expression on the tumor vascular endothelium and promotes an increase in T cell tumor invasion [116]. In the laboratory, administration of bevacizumab led to a decrease in MDSCs in the RCC mouse model, as well as a decrease in Tregs in vitro and in vivo [114,117]. A similar decrease in Tregs has been observed in RCC patients treated with sunitinib, where it correlated with overall survival [118].

#### **8. Angiogenesis Inhibitors and Immunotherapy**

Although the immune system is very effective in inducing an immune response against foreign antigens, malignant tumors can avoid immune surveillance via multiple mechanisms of immune tolerance. Overexpression of immune checkpoint molecules inducing immune tolerance has been demonstrated in some solid tumors, and correlates with poor prognosis [119]. Programmed-cell death-1 (PD-1 is a checkpoint molecule expressed on the outer surface of NK cells, B cells, DCs, monocytes, and CD4 + and CD8 + T cells [120]. When PD-1 is expressed by T cell stimulation and binds to PD-L1 and PD-L2 on antigen-presenting cells and some cancer cells, the Ras/MAPK/ERK kinase/ERK pathway and PI3K/AKT pathway are inhibited and inactivate T cells [120]. PD-L1 is expressed in cancers of tissues such as the lung, colon, ovaries, as well as in malignant melanoma and its expression is enhanced by the inflammatory cytokine interferon gamma [121]. Additionally, activation of HIF-1 in a hypoxic environment within a tumor leads to elevated expression of PD-L1 in cancer tissue [122]. In other words, cancer cells escape immune surveillance by inactivating locally accumulated T cells through the PD-1/PD-L1 pathway. Immunological checkpoint inhibitors such as nivolumab and pembrolizumab, which are PD-1 inhibitors, and atezolizumab, which is a PD-L1 inhibitor, promote the antitumor activity of T cells by blocking these pathways, and are clinically effective in several cancer types [122]. As mentioned in the previous section, angiogenesis inhibitors and immunological checkpoint inhibitors are expected to have a combined immunostimulatory effect. Increased infiltration of CD4 + and CD8 + T cells, in addition to macrophages, into the tumor space and increased expression of PD-L1 in the tumor by co-administration of bevacizumab and sunitinib have been shown in the RCC mouse model [123]. Additionally, a decrease in MDSCs in tumor tissue and an increase in PD-1 expression in tumor infiltrating lymphocytes have been observed in RCC patients treated with sunitinib [124]. Combination therapy with atezolizumab and bevacizumab resulted in an increase in CD8 + T cells and major histocompatibility complex 1 in the tumor, as well as up-regulation of chemokines and down-regulation of genes associated with neovascularization in patients with metastatic RCC [125]. Several phase III studies on the combined treatment of angiogenesis inhibitors and immunity checkpoint inhibitors are in progress, and the results of these preclinical and clinical trials are listed in Table 3. Two phase III trials have shown that a combination therapy of atezolizumab and bevacizumab is effective and tolerable. Comparisons of combination chemotherapies (carboplatin and paclitaxel) and atezolizumab + bevacizumab in untreated non-small cell lung cancer have shown better survival (response rate, progression-free survival, and overall survival) in the atezolizumab + bevacizumab group than in the chemotherapy + bevacizumab group. Subgroup analysis of the low PD-L1 expression group, the group with low effector T cell gene expression, and the liver metastases group also shows similar results [126]. In a study of patients with metastatic RCC characterized by ≥1% PD-L1 expression, the combination therapy (bevacizumab and atezolizumab) group had a longer progression-free survival than the sunitinib monotherapy group [127].



### **9. Conclusions**

In this review, we have discussed the mechanism of tumor angiogenesis as well as antiangiogenic therapy from the perspective of the tumor microenvironment. Although angiogenesis inhibitors have been used in combination with chemotherapy for more than 10 years, resulting overall survival has increased by only a few months and resistance to treatment has often developed rapidly. Angiogenesis inhibitors have failed to improve overall survival in some cancers such as breast cancer. These findings highlight the complexity of the pathways involved in tumor neovascularization and raise questions about the effective use of antiangiogenic therapy in cancer treatment. Therefore, we need to better understand the role of neovascularization in different cancers and how they avoid the effects of antiangiogenic therapy. A combination therapy with angiogenesis inhibitors and immunotherapy effectively enhances the benefits of angiogenesis inhibitors and represents the most promising path ahead.

**Author Contributions:** Y.K. (Yuki Katayama): Writing—Original draft preparation, J.U.: Writing—review & editing; Y.C., N.T., Y.K. (Yoshiko Kaneko) and T.Y.: Editing review; K.T.: Supervision.

**Acknowledgments:** We would like to thank Editage (www.editage.jp) for English language editing.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


Cells and Enhances the Efficacy of a GM-CSF-Secreting Cancer Immunotherapy. *Clin. Cancer Res.* **2006**, *12*, 6808–6816. [CrossRef] [PubMed]


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Review*

### **Therapeutic Monoclonal Antibodies and Antibody Products: Current Practices and Development in Multiple Myeloma**

### **Francesca Bonello, Roberto Mina, Mario Boccadoro and Francesca Gay \***

Myeloma Unit, Division of Hematology, University of Torino, Azienda Ospedaliero-Universitaria Città della Salute e della Scienza di Torino, 10126 Torino, Italy; francesca.bonello@edu.unito.it (F.B.); roberto.mina.rm@gmail.com (R.M.); mario.boccadoro@unito.it (M.B.)

**\*** Correspondence: fgay@cittadellasalute.to.it; Tel.: +39-011-6333-4279/4301; Fax: +39-011-6333-4187

Received: 8 November 2019; Accepted: 16 December 2019; Published: 19 December 2019 -

**Abstract:** Immunotherapy is the latest innovation for the treatment of multiple myeloma (MM). Monoclonal antibodies (mAbs) entered the clinical practice and are under evaluation in clinical trials. MAbs can target highly selective and specific antigens on the cell surface of MM cells causing cell death (CD38 and CS1), convey specific cytotoxic drugs (antibody-drug conjugates), remove the breaks of the immune system (programmed death 1 (PD-1) and PD-ligand 1/2 (L1/L2) axis), or boost it against myeloma cells (bi-specific mAbs and T cell engagers). Two mAbs have been approved for the treatment of MM: the anti-CD38 daratumumab for newly-diagnosed and relapsed/refractory patients and the anti-CS1 elotuzumab in the relapse setting. These compounds are under investigation in clinical trials to explore their synergy with other anti-MM regimens, both in the front-line and relapse settings. Other antibodies targeting various antigens are under evaluation. B cell maturation antigens (BCMAs), selectively expressed on plasma cells, emerged as a promising target and several compounds targeting it have been developed. Encouraging results have been reported with antibody drug conjugates (e.g., GSK2857916) and bispecific T cell engagers (BiTEs®), including AMG420, which re-directs T cell-mediated cytotoxicity against MM cells. Here, we present an overview on mAbs currently approved for the treatment of MM and promising compounds under investigation.

**Keywords:** multiple myeloma (MM); immunotherapy; monoclonal antibodies (mAbs); antibody products; B cell maturation antigens (BCMAs); bispecific T cell engagers (BiTEs®)

#### **1. Introduction**

Multiple myeloma (MM) is a hematologic malignancy characterized by a clonal expansion of aberrant plasma cells in the bone marrow inducing bone lesions, anemia, renal insufficiency and hypercalcemia. In the last two decades, the treatment armamentarium of effective anti-myeloma drugs, used both at diagnosis and at relapse, has been significantly expanded with various compounds of different drug-classes. However, despite the availability of several treatment options, MM still remains an incurable disease whose natural history is characterized by phases of disease remission followed by relapses. The remission duration tends to progressively decrease at every subsequent relapse and MM inevitably becomes refractory to all available agents. Therefore, even if the survival of MM patients, both young and elderly, has steadily increased over time, to date, roughly 50% of patients are alive at 5 years after diagnosis [1–3].

With the introduction of effective novel agent combinations, based on immunomodulatory agents (IMiDs) and proteasome inhibitors (PIs), the treatment goal for first-line therapies is now the achievement of minimal residual disease (MRD) negativity [4], which is currently reported in

50–80% of transplant-eligible [5–8] and in 15–30% of transplant-ineligible patients [9–11]. A large meta-analysis demonstrated that reaching MRD negativity (though with some variability in terms of methods and cut-offs adopted) significantly prolonged progression-free survival (PFS) and overall survival (OS) as compared to a MRD-positive status [12]. For this reason, efforts should be made to improve the effectiveness of first-line therapies in inducing deep and durable responses. Regardless of the effectiveness of newer combinations available at diagnosis, the prognosis of high-risk patients (e.g., patients with unfavorable genetics or molecular abnormalities, International Staging System (ISS) stage III, extramedullary disease, or those who experience an early relapse after first-line therapies) is dismal compared to that of standard-risk patients. This evidence prompts the development of different strategies and the adoption of newer drugs in this population, currently representing an unmet medical need.

Furthermore, despite the depth of response obtained with first-line therapies and the duration of the remission, relapse is inevitable in almost all patients with MM, who progressively become refractory to all approved drugs, particularly to IMiDs and PIs. The development of compounds with different mechanisms of action, aiming at synergizing with currently used agents and overcoming drug-induced resistance, is therefore a priority.

Immunotherapy, either passive—with monoclonal antibodies (mAbs) or cellular products directed against neoplastic cells—or active—when the patient's immune system is stimulated to mount an immune response against tumor cells—represents a pivotal strategy for the treatment of both solid and hematologic malignancies.

MAbs have entered the clinical practice for the treatment of MM [13]. They are selective compounds targeting surface antigens that are highly expressed on aberrant plasma cells and not (or at low density) on normal tissues, thus promoting on-target activity while limiting off-target toxicity. MAbs elicit their therapeutic actions through different mechanisms, including a direct cytotoxicity on the neoplastic cell and immune-mediated mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). Monoclonal antibodies can also be exploited to directly target the myeloma cell while conveying a cytotoxic agent, as in the case of antibody-drug conjugates (ADCs), or to engage and activate T cells against the myeloma cell as with bispecific T cell engagers.

Several potential targets have been identified on the myeloma cells and likewise constructs have been designed and tested in MM patients, some of them having already entered the clinical practice.

This review focuses on the strength and controversies of the current treatment strategies exploiting mAbs in MM, as well as on newer experimental immunotherapeutic approaches such as ADCs and bispecific T-cell engagers (BiTEs®).

#### **2. Monoclonal Antibodies**

#### *2.1. Anti-CD38 Monoclonal Antibodies*

#### 2.1.1. Rationale

CD38 is a transmembrane type II glycoprotein that is highly expressed on normal plasma cells as well as on MM cells [14]. CD38 is also present at lower levels on normal lymphoid and myeloid cells, on red blood cells, as well as on solid tissues such as muscle cells (especially in the airway system), epithelial cells in the prostate and pancreatic beta cells. CD38 acts as a receptor, as an adhesion molecule, and as an ectoenzyme [15–17].

Anti-CD38 mAbs elicit their action targeting CD38+ MM cells and inducing effector mechanisms such as ADCC (which relies mainly on natural killer [NK] cells), ADCP, and CDC [18–21]. An in vitro comparison between the different anti-CD38 molecules showed that ADCC was equally induced by all of them, whereas daratumumab induced the highest CDC at low concentration and ADCP [22]. Alongside immune-mediated cytotoxicity, anti-CD38 mAbs have an immunomodulatory activity that relies on the modulation of immune cells. Myeloid-derived suppressor cells (MDSC), regulatory B cells

(Bregs, which promote tumor growth and immune escape), as well as a subset of regulatory T cells (Tregs) express CD38, and their levels are reduced after daratumumab exposure. Conversely, daratumumab results in significant expansion of CD8+ cytotoxic and CD4+ helper T cells, likely following the depletion of regulatory cells. Remarkably, expanded effector T cells also show increased killing capacity due to augmented levels of granzyme B, which activates caspases and triggers cell apoptosis [23–25]. In addition, among the activities promoted by CD38, there is a nicotinamide adenine dinucleotide (NAD)-ase activity, which results in reduced levels of NAD+ in T cells, responsible for the loss of their effector functions (exhausted T cells). In murine models, anti-CD38 mAbs administration induced higher levels of NAD+ in T effector cells, thus enhancing their antitumor activity [23]. The main mechanisms of action of anti-CD38 monoclonal antibodies are summarized in Figure 1.

**Figure 1.** Main mechanisms of action of anti-CD38 monoclonal antibodies. Abbreviations: MDSC, myeloid-derived suppressor cell; ADCC, antibody-dependent cell-mediated cytotoxicity; CDC, complement-dependent cytotoxicity; NAD, nicotinamide adenine dinucleotide; ADPR, adenosine ribose; MAC, membrane attack complex.

In vitro studies showed a marked synergism between anti-CD38 mAbs (daratumumab, isatuximab and MOR202) and IMiDs (lenalidomide and pomalidomide), mainly owing to an enhanced NK activity elicited by IMiDs that increases both number and activity of NK cells and consequently ADCC, as well as the cytotoxic activity of macrophages, thus stimulating ADCP [26]. This evidence prompted the investigation of the in vivo effect of the addition of anti-CD38 mAbs to IMiD-based combinations. Anti-CD38 mAbs also show an additive effect with PIs [27], although the exact mechanisms are less clear.

#### 2.1.2. Clinical Development

#### Daratumumab

Daratumumab was the first fully human anti-CD38 mAb to be tested in clinical trials. Results of the main clinical trials are summarized in Table 1.


**Table 1.** Results of the main clinical trials with anti-CD38 monoclonal antibodies daratumumab and isatuximab.

Abbreviations: ORR, overall response rate; PFS, progression-free survival; OS, overall survival; Dara, daratumumab; Isa, isatuximab; V, bortezomib; C, cyclophosphamide; d, dex, dexamethasone; T, thalidomide; R, lenalidomide; K, carfilzomib; Poma, pomalidomide; M, melphalan; P, prednisone; NR, not reached; NA, not yet available; TNE, transplant ineligible; TE, transplant eligible; CR, complete response.

In the phase I GEN501 study, which investigated different doses of daratumumab in relapsed/ refractory (RR)MM patients, the greatest activity was reported at the dose of 16 mg/kg, at which 36% of patients achieved a partial response (PR) or better. These results were confirmed by the phase II SIRIUS trial, which reported a 29% overall response rate (ORR) in heavily pretreated patients, resulting into median PFS and OS of 3.7 and 17.5 months, respectively [41]. These results led to the approval, by both the Food and Drug Administration (FDA) and the European Medicines Agency (EMA), of daratumumab as single agent for RRMM patients with 3 prior lines of therapy including a PI and an IMiD.

The synergism showed in vitro by daratumumab and lenalidomide was first translated into a marked in vivo activity of the 3-drug combination daratumumab-lenalidomide-dexamethasone (Dara-Rd) observed in RRMM patients enrolled in the phase II GEN503 study and then confirmed by the phase III POLLUX study. In the POLLUX trial, 569 RRMM patients were randomized to receive standard lenalidomide-dexamethasone (Rd) versus Dara-Rd until disease progression or intolerance [29]. ORR was higher in the triplet arm (93% vs. 76%) as well as the rate of patients achieving minimal residual disease (MRD) negativity (26% vs. 6% of patients, threshold 10−<sup>5</sup> ). Median PFS was not reached (NR) versus

17.5 months (hazard ratio [HR] 0.41, *p* < 0.001) in the Dara-Rd versus Rd arms; this benefit was also consistent in patients with high-risk cytogenetics (HR 0.53, *p* = 0.09) [42]. Of notice, the addition of daratumumab to Rd did not significantly increase the rates of grade 3–4 toxicities, with the exception of neutropenia (54% vs. 39%) and infections (28.3% vs. 22.8%). These data supported the approval of Dara-Rd for the treatment of MM patients who had previously received at least 1 line of therapy.

Daratumumab was then evaluated with pomalidomide and dexamethasone (Dara-Pd). In a preliminary phase II trial, this 3-drug regimen showed, in a heavily pretreated population (the median number of prior therapies was 4), ORR (60%) and median PFS (8.8 months) that compared favorably with those of Pd alone (ORR 31%, median PFS 3.8 months) despite the limitations of a cross-trial comparison [32,43]. Following the results of this study, the triplet Dara-Pd received accelerated approval by the FDA for RRMM patients who previously received both an IMiD and a PI. This combination is appealing considering that, in the near future, the majority of newly diagnosed (ND)MM patients will become refractory to continuous lenalidomide after their first line of therapy. Definitive results will come from the phase III trial APOLLO (NCT03180736) comparing Dara-Pd vs. Pd in RRMM patients.

Daratumumab has also been associated with PIs. The phase III CASTOR trial compared bortezomibdexamethasone (Vd) administered for 8 cycles to daratumumab-Vd (Dara-Vd) for 8 cycles, followed by monthly daratumumab until progression in RRMM patients [30]. The addition of daratumumab resulted in higher ORR (83% vs. 63%) and MRD negativity rate (12% vs. 2%, threshold 10−<sup>5</sup> ), and in prolonged PFS (median, 16.7 vs. 7.1 months; HR 0.31; *p* < 0.0001) [31]. Importantly, the MRD negativity rate continued to increase over time for patients receiving Dara-Vd as compared to those receiving Vd, thus highlighting the benefit of continuous treatment with daratumumab. The PFS advantage was also consistent for patients previously exposed to bortezomib (HR 0.35, *p* < 0.001) and for patients with high-risk cytogenetic features detected by fluorescence in situ hybridization (FISH, HR 0.45, *p* = 0.05). The triplet Dara-Vd is currently approved by the FDA and EMA for RRMM patients.

A phase Ib study with carfilzomib-dexamethasone-daratumumab (KdD) induced an objective response in 84% of RRMM patients after both lenalidomide and bortezomib [33]. It was recently announced that the phase III CANDOR study (NCT03158688) comparing Kd to KdD met its primary endpoint, with a 37% reduction in the risk of progression or death (HR 0.63, 95% CI 0.464–0.854, *p* = 0.0014) in patients receiving daratumumab [44].

Because CD38 expression is higher in the early stages of the disease, and mAbs greatly rely on the immune system to exploit their anti-MM activity, it seems reasonable to expect that moving daratumumab to the first-line setting, when the immune-system of a treatment-naïve patient is less compromised, could increase its efficacy. In older patients with newly diagnosed (ND)MM, daratumumab plus bortezomib-melphalan-prednisone (Dara-VMP), followed by daratumumab maintenance, significantly increased the MRD negativity rate as compared to the standard of care VMP (22% vs. 6%, *p* < 0.001, threshold 10−<sup>5</sup> ), ultimately prolonging the median PFS (NR after a median follow-up of 17 months vs. 18.1 months, HR 0.50, *p* < 0.001) [11]. Highlighting the role of continuous treatment, a substantial benefit in PFS was detected during the maintenance phase when a lower rate of relapses was observed in patients receiving daratumumab compared with observation (sustained response after 18 months: 77% vs. 60%). This evidence supports the benefit of continuous therapy with daratumumab, which allows better disease control over time compared to fixed duration treatment. A longer follow-up is needed to detect an OS benefit. Dara-VMP has recently been approved by both the FDA and EMA, thus becoming one of the standards of care for transplant-ineligible patients. Impressive results in terms of higher MRD negativity rates (24.2% vs. 7.3%, respectively; *p* < 0.001, threshold 10−<sup>5</sup> ) and reduced risk of progression or death (median NR vs. 32 months after a median follow-up of 28 months, HR 0.56, *p* < 0.001) were observed when Dara-Rd was compared to Rd in NDMM patients not suitable for autologous stem-cell transplantation (ASCT; MAIA study [38]). In both Dara-VMP and Dara-Rd regimens, the addition of daratumumab did not negatively affect the safety profiles of VMP and Rd, despite a higher rate of grade 3–4 infections being reported in both studies in patients receiving daratumumab (Dara-VMP 23.1% vs. VMP 14.7%; Dara-Rd 32% vs. Rd

23%); also, the frequency of grade 3–4 neutropenia was higher in patients receiving daratumumab in the MAIA study (50% vs. 35%).

Daratumumab has also been incorporated in the induction, consolidation, and maintenance approach in combination with standard triplets such as bortezomib-thalidomide-dexamethasone (VTd) and bortezomib-lenalidomide-dexamethasone (VRd) as initial treatment for NDMM patients eligible for high-dose melphalan and ASCT.

The phase III CASSIOPEIA trial randomized 1085 transplant-eligible patients to VTd with or without daratumumab as induction and consolidation, followed by daratumumab maintenance or no maintenance. After the consolidation phase, the proportion of MRD-negative patients was higher in the Dara-VTd group than in the VTd group (64% vs. 44%, *p* < 0.001, threshold 10−<sup>5</sup> ). This translated into a significantly reduced risk of progression or death in the Dara-VTd arm as compared to the control group (HR for PFS 0.47, *p* < 0.001) [39]. The higher MRD negativity rate reported with daratumumab was also confirmed in ISS-III and high-risk FISH patients (64% vs. 46%, *p* = 0.01; 60% vs. 40%, *p* = 0.06, respectively), with a trend towards PFS improvement with daratumumab (HR 0.66, 95% CI 0.31–1.39; HR 0.67, 95% CI 0.35–1.30, respectively) in these subsets of patients that traditionally represent unmet clinical needs [45]. Mobilization and stem collection after a more intensified induction including daratumumab were adequate. Although patients in the Dara-VTd arm required the use of plerixafor more frequently (22% vs. 8%) and collected less CD34+ cells (median 6.3 × 10<sup>6</sup> /kg vs. 8 × 10<sup>6</sup> /kg), successful ASCT and hematopoietic reconstitution were not affected. Data on maintenance are eagerly awaited. Following the results of the CASSIOPEIA trial, in September 2019, the FDA has approved frontline Dara-VTd as induction for transplant-eligible patients. VRd ± daratumumab as induction and post-ASCT consolidation followed by lenalidomide ± daratumumab maintenance is being compared in the ongoing phase II GRIFFIN trial [40]. The quadruplet significantly improved the MRD negativity rate (threshold 10−<sup>5</sup> ) at the end of consolidation, as compared to VRd (47.9% vs. 17.9%, HR 0.23, *p* < 0.001). In both trials, patients treated with daratumumab experienced no significant increase in grade 3–4 non-hematologic adverse events (AEs). Data on maintenance will shed light on the role of daratumumab maintenance, either alone or in combination with lenalidomide.

Other ongoing phase II/III trials evaluating front-line daratumumab in ASCT-eligible patients include the EMN17/PERSEUS trial, which explores the addition of daratumumab to VRd as induction and consolidation and to lenalidomide as maintenance treatment, and the EMN18 study, which compares induction and consolidation with daratumumab-bortezomib-cyclophosphamidedexamethasone (Dara-VCd) to standard VTd followed by ASCT and maintenance with ixazomib ± daratumumab [46]. The main ongoing trials are summarized in Table 2.


**Table 2.** Main ongoing trials involving daratumumab and isatuximab in multiple myeloma patients.


**Table 2.** *Cont.*

Abbreviations: pts, patients; NDMM, newly diagnosed multiple myeloma; RRMM, relapsed/refractory MM; Dara, daratumumab; Isa, isatuximab; ASCT, autologous stem-cell transplantation; TE, transplant eligible; NTE, transplant ineligible; Ixa, ixazomib; V, bortezomib; C, cyclophosphamide; d, dex, dexamethasone; T, thalidomide; R, lenalidomide; K, carfilzomib; Poma, pomalidomide.

It is currently a matter of debate whether patients with smoldering (S)MM should receive therapy with the aim of preventing the progression to symptomatic MM and the associated morbidity. Two randomized trials demonstrated the benefit of lenalidomide, with or without dexamethasone, in delaying the time to progression to active MM versus observation; importantly, the longer follow-up of the Spanish trial allowed for the detection of an OS advantage for lenalidomide-treated patients [51]. In this setting, a highly targeted therapy with a good safety profile stands out as an ideal option. In the phase II CENTAURUS trial, single-agent daratumumab resulted in an ORR of 56% in high-risk SMM patients, and median PFS was NR after a median follow-up of 26 months [52,53]. The randomized phase III AQUILA study is currently comparing daratumumab administered for 3 years versus standard observation in high-risk SMM (NCT03301220).

One of the limitations to the use of daratumumab is its long infusion time (3.5 h). To deal with this issue, a shorter infusion schedule was tested—daratumumab was administered over a 90 min infusion at the usual dose (16 mg/kg) from the third infusion onward, without increasing the risk for infusion-related reactions (IRRs) or further short-term AEs [54]. A game changer in this setting will be the possibility of delivering daratumumab subcutaneously over a short period of time. The PAVO study explored subcutaneous daratumumab in combination with the recombinant human hyaluronidase PH20 enzyme (rHuPH20), which allowed for the reaching and maintaining of a high-serum concentration of

the mAb [55]. At the end of phase Ib of the study, a flat dose of 1800 mg was recommended on the basis of pharmacokinetics, safety (all-grade IRRs 25%), and efficacy data (ORR 42%).

#### Isatuximab

Isatuximab (SAR 650984) is an anti-CD38 immunoglobulin G (IgG)-k chimeric monoclonal antibody that, besides having the same mechanisms of action of daratumumab, holds a unique direct proapoptotic effect independent from the Fc cross-linking [56,57]. Results of the main clinical trials are summarized in Table 1.

Similarly to daratumumab, isatuximab showed a promising activity when administered as a single agent in heavily pre-treated MM patients [58] and has therefore been combined with different anti-MM compounds. A phase Ib trial combined isatuximab at different dose levels with Rd in heavily pretreated MM patients (5 median prior lines of therapy), of whom 68% had already received carfilzomib or pomalidomide and 82% were refractory to lenalidomide. ORR was 51% (and 52% in lenalidomide-refractory patients) and median PFS was 8.5 months. IRRs were the most common AEs related to isatuximab (56% of patients, mainly of grades 1–2 and limited to first infusions) [34]. Another phase Ib trial combined isatuximab with Pd in relapsed patients (3 median prior lines of therapy)—the ORR was 62% and the median PFS was 17.6 months [35]. For both combinations, the selected dose of isatuximab was 10 mg/kg for 4 weekly doses and every 2 weeks thereafter. Of notice, preliminary results of a phase Ib trial in which isatuximab was combined to Kd showed a promising 66% ORR [36].

The ongoing phase III ICARIA trial (NCT02990338) is comparing the triplet isatuximabpomalidomide-dexamethasone (Isa-Pd) to Pd in 307 RRMM patients who had received at least 2 previous lines of therapy (median lines: 3 in both groups). After a median follow-up of 11.6 months, a consistent benefit in terms of ORR (60% vs. 35%, *p* < 0.001) and PFS (median PFS 11.5 vs. 6.5, HR 0.59, *p* = 0.001) for the triplet arm compared to the control group was shown. Subgroup analysis revealed that PFS benefit was also maintained in high-risk patients (median PFS 7.5 vs. 3.7 months, HR 0.66, 95% CI 0.30–1.28). The median OS was NR in either group, although a trend to improved OS was observed in the triplet arm (HR 0.687, 95% CI 0.461–1.023; *p* = 0.06) [37]. Regarding the safety profile, Isa-Pd induced a slightly higher rate of grade 3–4 infections (42.8% vs. 30.2%) and neutropenia (84.9% vs. 70.1%) [50]. The ongoing phase III IKEMA trial is evaluating the combination of isatuximab with Kd in RRMM patients (NCT03275285).

In transplant-ineligible NDMM patients, isatuximab (10 mg/kg) is being evaluated in a phase Ib trial in combination with VRd as induction (4 cycles) followed by maintenance with Isa-Rd. Preliminary results showed an ORR of 93%, with 38.5% of patients achieving MRD negativity [47]. Another phase Ib trial is evaluating induction with 12 cycles of isatuximab (10/20 mg/kg) plus bortezomib-cyclophosphamide-dexamethasone (VCd), followed by maintenance with single-agent isatuximab in a similar patient population. The ORR was 87%, whereas data on MRD status and PFS are not yet available [48].

In order to improve the poor prognosis of high-risk patients, a phase Ib trial that was specifically designed for high-risk NDMM patients is currently testing a quadruplet regimen combining isatuximabcarfilzomib-lenalidomide-dexamethasone (GMMG-CONCEPT trial [59]).

Finally, the phase III IMROZ study is currently comparing the quadruplet isatuximab-VRd (Isa-VRd) to VRd as upfront treatment for transplant-ineligible patients (NCT03319667). Another ongoing trial is comparing the quadruplet Isa-VRd to isatuximab-VCd (Isa-VCd) in transplant-ineligible patients at diagnosis (NCT02513186).

#### MOR202 and TAK-079

MOR202 and TAK-079 are two anti-CD38 mAbs under development. In preliminary trials, MOR202 proved to be effective in combination with IMiDs; as expected, as this agent does not seem to induce CDC, a low rate of IRRs was observed (10%) [60,61]. In detail, the ORR was 28% in patients receiving MOR202 plus dexamethasone, which increased up to 65% in those receiving MOR202 plus

Rd and to 43% in those receiving MOR202 with Pd. However, further development of MOR202 has been discontinued in the United States and Europe. Subcutaneous TAK-079 is currently being tested in preliminary clinical trials on RRMM patients as monotherapy (NCT03439280) and in combination with standard regimens Rd or VRd (NCT03984097). We still need to further define the role of newer anti-CD38 mAbs in the treatment scenario for MM, where daratumumab and isatuximab have proven high efficacy and manageability.

#### *2.2. Anti-Signaling Lymphocytic Activation Molecule Family 7 (SLAMF7) Monoclonal Antibodies*

#### 2.2.1. Rationale

Signaling lymphocytic activation molecule family 7 (SLAMF7 or CS1) is a cell surface glycoprotein whose expression is essentially restricted to NK cells and both normal and abnormal plasma cells, with 95% of myeloma plasma cells being SLAMF7-positive [62]. In plasma cells and MM cells, the SLAMF7 pathway promotes cell growth and survival, as well as the interaction with the bone marrow micro-environment. Its highly selective expression on plasma cells makes SLAMF7 an optimal target for mAbs.

#### 2.2.2. Clinical Development

Elotuzumab is a humanized IgG-1 monoclonal antibody targeting SLAMF7 that promotes NK-mediated ADCC, directly activates NK cells and interferes with the MM cell adhesion to the bone marrow stromal cells [63–65]. Elotuzumab showed no clinically meaningful activity when administered as a single agent—in a phase I dose-escalating study, the best response achieved by RRMM patients treated at different doses of elotuzumab was stable disease (SD, 26%) [66] (Table 3).


**Table 3.** Results of the main clinical trials with anti-signaling lymphocytic activation molecule family 7 (SLAMF7) monoclonal antibody elotuzumab.

Abbreviations: Elo, elotuzumab; ORR, overall response rate; PFS, progression-free survival; OS, overall survival; d, dex, dexamethasone; R, lenalidomide; Poma, pomalidomide; V, bortezomib; NR, not reached; NA, not yet available.

Preclinical data showed a synergistic activity of elotuzumab with IMiDs, the latter altering cytokine production and enhancing the activity of NK cells, the main target of elotuzumab immune activity. Promising results in terms of efficacy and tolerability were observed combining elotuzumab with Rd in phase I and II studies, thus providing the rationale for the phase III study ELOQUENT-2, which compared elotuzumab-Rd (Elo-Rd) to Rd in RRMM patients that were not refractory to lenalidomide [67,68,72]. In this study, elotuzumab was administered at the dose of 10 mg/kg and treatment was continued until progression or intolerance [66]. The triplet regimen containing elotuzumab proved to be more effective than Rd in terms of both PFS (19.4 vs. 14.9 months, HR 0.70, *p* < 0.001) and OS (48 vs. 40 months), without adding significant toxicity [73,74]. Patients at first relapse after a remission duration >3.5 years obtained the greater PFS advantage with Elo-Rd [75], showing that the greatest benefit with Elo-Rd could be obtained in patients with a slow and indolent

progression. Elo-Rd is currently approved by both the FDA and EMA for the treatment of RRMM patients after 1 line of therapy.

The synergistic activity between elotuzumab and IMiDs prompted the investigators to test elotuzumab both in the upfront setting in combination with lenalidomide and at relapse with the third-generation IMiD pomalidomide [76]. The ongoing phase III study ELOQUENT-1, whose results are not yet available, enrolled NDMM patients ineligible for high-dose melphalan and ASCT in order to investigate the benefit of the addition of elotuzumab to the standard doublet Rd, possibly establishing a new standard of care in this setting.

In the randomized phase II ELOQUENT-3 trial, the addition of elotuzumab to Pd in RRMM patients significantly increased the ORR (53% vs. 26%) and prolonged median PFS (10.3 vs. 4.7 months, HR 0.54, *p* = 0.008), as compared to Pd alone. Again, the safety profiles of the two arms of the study were overlapping, meaning that elotuzumab did not add significant toxicity to Pd [69]. On this basis, in 2018 the FDA approved the triplet elotuzumab-Pd for the treatment of RRMM patients who had received at least 2 prior regimens including lenalidomide and a PI.

In preclinical models, elotuzumab activity was potentiated by bortezomib, which makes myeloma cells more vulnerable to NK-mediated lysis [77]. This combination was subsequently tested in clinical trials. In a phase I study on RRMM patients, elotuzumab was combined with bortezomib, showing an ORR of 48% and a median time to progression of 9.5 months [70]. The triplet elotuzumab-Vd (Elo-Vd, with elotuzumab administered at 10 mg/kg) was subsequently compared to Vd in a phase II trial on 152 RRMM patients, half of which had already received bortezomib in previous lines of therapy. ORR was similar between the two groups (66% vs. 63%), and a slight PFS advantage was observed in the triplet arm that nonetheless did not reach statistical significance (median, 9.7 vs. 6.9 months, HR 0.72, *p* = 0.09) [71]. The most common grade ≥3 AEs were infections (Elo-Vd 21% vs. Vd 13%) and thrombocytopenia (Elo-Vd 9% vs. Vd 17%). Because elotuzumab elicits its action by binding its Fc portion to the Fc gamma receptor III on NK cells, different allelic variants of the receptor were analyzed to evaluate possible predictors of elotuzumab efficacy. In this study, patients homozygous for the high-affinity Fc gamma receptor IIIa (FcγRIIIa) V allele showed longer PFS as compared to patients homozygous for the low-affinity allele. Considering the number of treatment options currently approved, the availability of a predictor of response could help clinicians in the choice of the most appropriate treatment.

Elotuzumab-Rd has also been investigated as a prevention strategy in high-risk SMM. In a phase II study (NCT02279394), patients received 8 cycles of elotuzumab-Rd and were subsequently allowed to continue with elotuzumab and lenalidomide maintenance until progression to symptomatic MM [78]. Preliminary data showed an ORR of 84% with no patients progressing at MM at the present follow-up of 29 months. Grade 3–4 toxicities included neutropenia (16%) and infections (12%), mainly related to lenalidomide. Again, single-agent elotuzumab did not show any clinical activity when in the setting of SMM [79].

Of interest, the rate of IRRs observed with elotuzumab—which were mostly mild in nature (grades 1–2) and rarely leading to treatment discontinuation—was definitely lower (10%) than that observed with other mAbs, making elotuzumab-based combinations appealing options for the treatment of frail patients [80].

Numerous studies are currently ongoing with elotuzumab-based combinations, such as elotuzumab-VRd (Elo-VRd, NCT02375555), elotuzumab-KRd (Elo-KRd, NCT02969837) and elotuzumab plus pomalidomide-bortezomib-dexamethasone (NCT02718833).

#### *2.3. Anti-Programmed Death 1 (PD-1) Monoclonal Antibodies*

#### 2.3.1. Rationale

The programmed death 1 (PD-1) receptor is a transmembrane glycoprotein expressed on antigen-activated T cells and B cells. The binding of PD-1 ligands (PD-1-L1 and PD-1-L2) on PD-1 receptor results in the downregulation of immune T cell functions [81]. Preclinical data showed that PD-1/L1 is highly expressed on myeloma cells and, at variable levels, on normal plasma cells. It is also expressed at high levels on dendritic cells in the myeloma microenvironment [82,83]. Moreover, T cells derived from myeloma patients showed higher rates of PD-1 expression as compared to T cells from healthy donors, suggesting that the PD-1/PD-L1 pathway plays an important role in the immune escape of myeloma cells. Given these premises, targeting PD-1 and PD-L1 with monoclonal antibodies seems to be a promising strategy for the treatment of MM.

#### 2.3.2. Clinical Development

Monoclonal antibodies directed against the PD-1/PD-L1 pathway can be divided into molecules targeting PD-1 (e.g., pembrolizumab and cemiplimab) and molecules targeting PD-L1 (e.g., durvalumab). Pembrolizumab monotherapy did not show efficacy as a single agent in 30 heavily pretreated myeloma patients (4 median prior lines of therapy) [84]. Pembrolizumab was subsequently combined with immunomodulatory agents, as preclinical data suggested that IMiDs could contribute to the downregulation of the PD-1/PD-L1 pathway [85]. In phase II trials, ORR was 50% in RRMM patients receiving pembrolizumab plus Rd and 60% in patients receiving pembrolizumab plus Pd [86]. However, in 2017, following the preliminary results of the two randomized phase III trials KEYNOTE 185 (pembrolizumab-Rd vs. Rd) and KEYNOTE 183 (pembrolizumab-Pd vs. Pd), the FDA prompted the discontinuation of any further investigations of these combinations, in light of the increased risk of death for patients in the pembrolizumab group versus the control group (HR for OS in pembrolizumab-Pd vs. Pd 1.61; HR for OS in pembrolizumab-Rd vs. Rd 2.06) [87,88]. The main concern with this combination is indeed the increased risk of enhancing immune-mediated toxicity, resulting in various AEs, such as dermatologic, pulmonary, cardiac, gastrointestinal and hepatic toxicities. These results questioned the utility of anti-PD-1 mAbs in MM, at least in combination with IMiDs. Different molecules are currently under evaluation in combination with other agents. The anti-PD-1 cemiplimab is being evaluated in a phase I/II trial in combination with isatuximab (NCT03194867), whereas durvalumab is being tested in combination with daratumumab (NCT03000452). However, the future role of this class of molecules in the treatment of MM remains debated.

#### **3. Antibody Drug Conjugates**

#### *3.1. Rationale*

Antibody drug conjugates (ADCs) are monoclonal antibodies bound by a chemical linker to a cytotoxic compound directed against surface antigens of the targeted cells. ADCs selectively target cells expressing their target antigen and are then internalized releasing the cytotoxic component through lysosome degradation, causing cell death. This targeted delivery limits the systemic exposure to the cytotoxic compound, sparing the non-malignant cells and tissues that do not express the target antigen, consequently limiting its off-target toxic effects [89,90]. In the past few years, interest has been raised around ADCs for the treatment of lymphoid malignancies, with brentuximab vedotin being the first agent of this class to receive FDA and EMA approval for the treatment of relapsed/refractory Hodgkin lymphoma and anaplastic large cell lymphoma in 2011–2012 [91,92]. In MM, ADCs showed preclinical activity in in vitro and in xenograft models and are currently under evaluation in clinical trials for relapsed MM patients [93–95]. One of the main challenges with ADCs is the choice of the most appropriate surface antigens to be targeted, which should be highly expressed only on malignant cells and not on normal tissues. Several target antigens have been identified on plasma cells: CD56, CD138, CD74, Fc receptor-like 5 and B cell maturation antigen (BCMA) [96]; of these, CD56 is expressed only on MM cells, with no expression on normal plasma cells, whereas other antigens are expressed on both malignant and non-malignant plasma cells, although at different levels [97]. The cytotoxic compound is typically a small molecular weight toxin with potent activity at low concentrations. Such molecules, usually not employed for systemic chemotherapy due to their excessive toxicity, can cause cell death due

to two different mechanisms: cell cycle interference through microtubules inhibitions and DNA damage. Maytansinoid derivatives are microtubule inhibitors, including DM1 (emtansine and mertansine), DM4 (soravtansine and ravtansine) and auristatin derivatives (including monomethyl-auristatin E (MMAE, vedotin) and monomethyl auristatin F (MMAF, mafodotin)) [98–101]. Calicheamicins, duocarymycins and pyrrolobenzodiazepine dimmers are DNA-damaging agents [102,103].

#### *3.2. Clinical Development*

Table 4 summarizes the results of the main studies with ADCs in MM.


**Table 4.** Results of preliminary clinical trials with antibody-drug conjugates (ADCs).

Abbreviations: R, lenalidomide; d, dex, dexamethasone; ORR, overall response rate; PFS, progression-free survival; OS, overall survival; NR, not reached; CRS, cytokine release syndrome; G, grade; MMAF, monomethyl auristatin F; BCMA, B cell maturation antigen.

In 2018, the results of a first in-human phase I study investigating GSK2857916, a BCMA-targeting mAb conjugated to the antimitotic agent monomethyl auristatin F (MMAF), in 73 RRMM patients were published. BCMA, a transmembrane receptor required for B cell maturation, was chosen as an optimal target, as it is expressed almost exclusively on MM cells and plasma cells [104–106]. In the dose-escalation phase of the study, 38 patients received escalating doses of IV GSK2857916 (0.03–4.6 mg/kg) every 3 weeks. In the dose-expansion phase of the study, 35 patients received the recommended phase II dose of GSK2857916 (3.4 mg/kg) every 3 weeks until progression. Among heavily pre-treated patients, GSK2857916 induced an objective response in 60% of them, with 15% of patients achieving a CR or a stringent CR (sCR). Remarkably, the ORR in patients previously treated with anti-CD38 mAbs and refractory to both IMiDs and PIs was 38%. Responses were rapid (median time to response 1.2 months) and durable (median duration of response 14.3 months). Overall, median PFS was 12 months; median PFS was 7.9 months in double-refractory patients (to IMiDs and PIs) and 6.2 months in double-refractory patients with prior daratumumab. The most common treatment-related toxicities were thrombocytopenia (63%; grades 3–4: 26%) and corneal events in terms of blurred vision and photophobia (51%; grades 3–4: 3%). Ocular toxicity was mainly limited to grades 1–2 and was reversible and easily manageable with dose reductions (51% of patients) [104,106]. Because GSK285791 showed high ORR in patients previously treated with anti-CD38 mAbs, a phase I/II clinical trial exploring its efficacy as monotherapy in patients with previous exposure to daratumumab/isatuximab has recently completed enrollment and results will soon be available (NCT03525678). Ongoing trials are evaluating its safety and efficacy in combination with pembrolizumab (NCT03848845), pomalidomide (NCT03715478), and lenalidomide versus bortezomib (NCT03544281).

Indatuximab-ravtansine (BT062) is an anti-CD138 IgG4 monoclonal antibody that delivers the microtubule inhibitor maytansinoid ravtansine to CD138-positive cells. CD138 is a transmembrane protein receptor upregulated by myeloma cells. BT062 monotherapy was evaluated in 67 heavily pretreated RRMM patients (median previous therapies 7, range 1–15). The most common grade 3–4 toxicities were fatigue (7%), anemia (7%), and diarrhea (4%). At the maximum tolerated dose (MTD) of BT062 (140 mg/m<sup>2</sup> ), 62% of patients achieved SD, whereas an objective response was observed in 5% of patients only. Median PFS and OS were 3 and 26 months, respectively [107]. BT062 is currently under evaluation in combination with lenalidomide or pomalidomide plus dexamethasone in RRMM patients. In patients receiving BT062 + lenalidomide (*n* = 47), ORR was 77% and median PFS was 16.4 months, whereas in those receiving the ADC in combination with pomalidomide (*n* = 17) ORR was 79% and median PFS was NR after 7 months of follow-up. These triplets were well tolerated, with main AEs being fatigue and diarrhea [108].

Lorvotuzumab-mertansine (IMGN901) is an anti-CD56 mAb linked to the maytansinoid mertansine, which inhibits microtubules assembly interfering with cell cycle and therefore causing cell death. A phase I trial enrolling 37 heavily pre-treated patients (78% had ≥3 lines of therapy) with CD56+ RRMM explored the safety and efficacy of single-agent IMGN901. The MTD was established at 112 mg/m<sup>2</sup> . Forty-three percent of patients experienced SD, 6% PR, and no patient reached a very good (VG)PR or better, with a median PFS of 6.5 months. The toxicity profile was manageable and drug discontinuation due to AEs was observed in 24% of patients, with peripheral neuropathy (grades 3–4: 5.3%) being the most common toxicity leading to discontinuation [109]. IMGN901 is also being evaluated in combination with Rd. Preliminary reports showed an ORR of 56%, including 2 CRs and 8 VGPRs. The most common toxicity was peripheral neuropathy, although no grade 3–4 events occurred at the MTD of 75 mg/m<sup>2</sup> [111].

ADCs, particularly GSK285791, displayed a promising efficacy among heavily pre-treated patients. Their unique mechanism of action and preliminary efficacy data make these drugs an appealing treatment option in patients who have become refractory to IMiDs, PIs, and anti-CD38. Furthermore, the lack of cross-resistance with currently approved agents also prompts their investigation in the earlier phase of the disease, such as in the context of a consolidation strategy in high-risk patients or those MRD-positive after the induction/transplant phases.

Other compounds are under preliminary evaluation in MM. CD74, a transmembrane glycoprotein expressed in more than 90% of B cell malignancies, is the target of the ADC milatuzumab-doxorubicin (of hLL1-DOX) [110]. In a preliminary study, the ADC proved to be well tolerated, with SD being the best response achieved (26% patients) with this agent used as monotherapy for RRMM patients [112]. Preclinical results showing synergistic activity of hLL1-DOX with PIs and IMiDs provide the biological rationale for the evaluation of this ADC in combination with other agents.

#### **4. Bispecific T Cell Engagers**

Bispecific monoclonal antibodies are engineered molecules meant to redirect immune effector cells, mainly T and NK cells, to tumor cells, thus restoring the immune suppressor activity of the immune system against neoplastic cells. Bispecific T cell engager molecules are a class of bispecific antibodies combining the minimal binding domains (variable fragments (Fv), single chains) of two different monoclonal antibodies on one polypeptide chain [113]. They are characterized by a small size, allowing optimal proximity between the engaged T cell and the target tumor cell; for this very reason, they are active at low concentrations, as compared to bispecific antibodies. Bispecific antibodies usually link the invariant part of CD3 of the T cell receptor (TCR) on T cells and a tumor-specific antigen, thus leading to T cell activation and proliferation and tumor cell apoptosis [114]. The first approved bispecific T cell engager was the anti-CD19 blinatumomab for the treatment of RR B cell acute lymphoblastic leukemia [115].

Among potential targets on plasma cells, BCMA, CD38 and SLAMF7 have been chosen to design anti-MM bispecific antibodies [23,116], with BCMA representing the most promising target. Another potential target due to its high expression on PC is G-protein coupled receptor C family 5D (GPRC5D), whose function is still unclear [117,118].

### *Clinical Development*

AMG420 is an anti-BCMA bispecific T cell engager that is currently being evaluated in the first in-human dose escalation trial enrolling RRMM patients (4 median prior lines of therapy). AMG420 was administered as a continuous intravenous infusion due to its short half-life at doses ranging from 0.2 to 800 mcg/die. At the MTD of 400 mcg/day, the ORR was 70%, with 5/10 patients obtaining MRD-negative sCRs (10−<sup>4</sup> ) [119]. Dose-limiting toxicities were cytokine release syndrome (CRS, 1 patient) and peripheral neuropathy (2 patients). Only 1 grade 3 CRS was observed, and no grade 3–4 AEs related to the central nervous system were registered at the MTD. Another anti-BCMA BiTE®, AMG 701, has a longer half-life (112 h), thus allowing weekly short-term infusion. AMG 701 is currently being investigated in the first phase I trial [120].

BiTEs® currently under investigation are listed in Table 5.

**Table 5.** Bispecific T cell-engaging agents (BiTEs®) for the treatment of multiple myeloma.


Abbreviations: BCMA, B cell maturation antigen; CPRC5D, G-protein coupled receptor C family 5D.

#### **5. Conclusions and Future Directions**

Immunotherapy, and in particular mAbs, is no longer an appealing future perspective, but rather a valuable present therapeutic option for MM patients—having demonstrated to induce a response where conventional agents had failed—to increase the depth of response obtained with standard regimens acting in synergy with them and, ultimately, to prolong both PFS and OS. The 'guiding star' in this treatment landscape is definitely the anti-CD38 mAb, which rapidly turned from being a valid alternative for RRMM patients without further viable therapeutic options to being the backbone of virtually all present and future combinations adopted as frontline therapies. However, given the different combinations of both daratumumab and isatuximab with backbone therapies (available or under evaluation), we still need to define which anti-CD38 mAb should be used considering the unavailability of data on the superiority of one over the other. Another open issue is as to what could be the effectiveness of re-treatment with the same, or a different, CD38 mAb. Arguably, this last question will be answered in the near future, thanks to the increasing use of anti-CD38 mAb combinations in early lines. These issues are particularly challenging considering the wide heterogeneity of myeloma cell populations [121]. Immunotherapy seems to be a potential strategy for targeting virtually all tumor subclones, as effector mechanisms rely on the patient immune system. Ongoing studies are exploring the different potential mechanisms of resistance to anti-CD38 mAbs, as well as how to overcome them. Lower basal levels of the target antigen have been proposed as a possible mechanism of intrinsic resistance to mAbs [122,123]. Regarding daratumumab, the downregulation of CD38 on cell surfaces could partially explain the loss of response to mAb therapy [124]. Interestingly, myeloma cells exposed to isatuximab and MOR202 did not show such a downregulation [125,126]. An intriguing way to overcome the acquired resistance derived from antigen downregulation could be the addition of molecules able to re-induce CD38 expression on cell surface, such as all-trans retinoic acid (ATRA) or panobinostat [127,128]. Finally, other proposed mechanisms of resistance under evaluation include the modification of the expression of adhesion molecules and the overexpression of complement inhibitors. In the context of the currently available anti-CD38 combinations, the role

of anti-SLAMF7 mAb-based combinations is unclear—both anti-CD38 and anti-SLAMF7 antibodies have been combined with the same backbones (Rd, Pd, Vd); both anti-CD38 and anti-SLAMF7 mAbs showed encouraging efficacy data even in high-risk patients, but not substantial enough to suggest an ability to completely overcome their adverse prognoses; and, finally, both mAbs have a very good safety profile. Studies showing the better efficacy of one mAb combination over the other are currently lacking. The role of both mAbs in the treatment of SMM also needs to be defined—their good safety profiles make them good candidates for the treatment of a still asymptomatic disease, but their efficacy and the possibility to improve OS still need to be shown. ADCs and BiTEs® are fascinating constructs potentially able to either carry toxic compounds or redirect T cells against MM cells in a very specific way, thus limiting off-target toxicities. The preliminary results obtained with single-agent ADCs or BiTEs® in heavily pre-treated patients are by far exceeding expectations, especially if compared to the results obtained with the currently available single-agent drugs. Future studies will shed light on their role in the treatment of MM patients and on their efficacy when used earlier in the course of the disease; they will also explore how to improve their feasibility and treatment compliance, especially in relation to the continuous intravenous infusion characteristic of the BiTEs® evaluated in MM thus far. In this field, the compounds showing the most encouraging preclinical results are bispecific antibodies with extended half-life such as the anti-BCMAs AMG701 and PF3135, which would allow a weekly administration [129,130]. Moreover, we still need to decipher the exact mechanisms of resistance and how to revert them, as well as the best drug-partners to enhance their efficacy in different settings. We have to devise the proper antigen selection and payload choice that will be critical for their success in the treatment of MM. MAbs can also be conjugated with radioisotopes in order to increase the antitumor effect of the molecules. Daratumumab has been combined with different radionuclides (e.g., actinium-225), resulting in an increased tumoricidal effect besides its Fc-effector functions in preclinical models [131]. Bispecific pretargeted radiolabeled antibodies showed an even greater biodistribution to tumor cells and, in future, can represent an appealing approach for the treatment of MM, especially for heavily pretreated patients who usually remain sensitive to radiation [132]. Regarding the use of mAbs, another field of interest is the use of radiolabeled antibodies for imaging assessment with immuno-positron emission tomography (immuno-PET) [133]. Indeed, surface antigens expressed on myeloma cells could be a target for radiolabeled mAbs, which would allow highly specific tumor detection and precise response assessment. Daratumumab has already been labeled to different positron emitters showing excellent targeting in preclinical models [134–136]. With these premises, immuno-PET could represent a useful tool for imaging assessment and also for guiding treatment strategies, as this technique could potentially be used to predict the effectiveness of mAb therapy.

Another issue is timing, that is to say, the most appropriate phase of treatment or disease in which these different classes of drugs should be used—if at diagnosis, at the evidence of MRD persistence in an effort to eradicate a resistant clone, or at relapse once conventional treatments have failed. In a highly competitive setting, with few validated targets (CS1, CD38, BCMA) and many different technologies (ADC, BiTEs®, chimeric antigen receptor [CAR] T cells), both preclinical and clinical studies are critical to identify the most promising compounds. Along with the refinement of the existing drug regimens and treatment strategies and the development of new ones, a better understanding of the role of the immune system in the pathogenesis of MM will certainly be necessary.

**Author Contributions:** Substantial contributions to the conception or design, F.B., R.M., M.B., and F.G.; acquisition, analysis, or interpretation of data, F.B., R.M., M.B., and F.G.; first draft, F.B., R.M., and F.G.; supervision, M.B. and F.G.; critical revision for important intellectual content, F.B., R.M., M.B., and F.G.; final approval of the version to be published, R.M., F.B., M.B., and F.G.; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved, F.B., R.M., M.B., and F.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** F.B. declares no competing financial interests. R.M. has received honoraria from Amgen, Celgene, Takeda, and Janssen, and served on the advisory boards for Janssen. M.B. has received honoraria from Sanofi, Celgene, Amgen, Janssen, Novartis, Bristol-Myers Squibb, and AbbVie, and has received research funding from Sanofi, Celgene, Amgen, Janssen, Novartis, Bristol-Myers Squibb, and Mundipharma. F.G. has received honoraria from Amgen, Bristol-Myers Squibb, Celgene, Janssen, and Takeda, and served on the advisory boards for Amgen, Bristol-Myers Squibb, Celgene, Janssen, Roche, Takeda, and AbbVie.

#### **References**


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*Review*
