International Journal of

Molecular Sciences
Review

Current Targeted Therapy for Metastatic Colorectal Cancer
Tomokazu Ohishi 1,2, * , Mika K. Kaneko 3 , Yukihiro Yoshida 4 , Atsuo Takashima 5 , Yukinari Kato 3,6, *and Manabu Kawada 2
1

2

3

4

5

6

*

Citation: Ohishi, T.; Kaneko, M.K.;
Yoshida, Y.; Takashima, A.; Kato, Y.;

Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation,
18-24 Miyamoto, Numazu-shi 410-0301, Shizuoka, Japan
Institute of Microbial Chemistry (BIKAKEN), Laboratory of Oncology, Microbial Chemistry Research
Foundation, 3-14-23 Kamiosaki, Shinagawa-ku 141-0021, Tokyo, Japan
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan
Department of Thoracic Surgery, National Cancer Center Hospital, 5-1-1 Tsukiji,
Chuo-ku 104-0045, Tokyo, Japan
Department of Gastrointestinal Medical Oncology, National Cancer Center Hospital, Tsukiji,
Chuo-ku 104-0045, Tokyo, Japan
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan
Correspondence: ohishit@bikaken.or.jp (T.O.); yukinari.kato.e6@tohoku.ac.jp (Y.K.);
Tel.: +81-55-924-0601 (T.O.); +81-22-717-8207 (Y.K.)

Abstract: Colorectal cancer (CRC) is the third most common type of cancer and the second leading
cause of cancer deaths worldwide. Surgery or surgery plus radiotherapy and/or chemotherapy forpatients with metastatic CRC (mCRC) were accepted as the main therapeutic strategies until the early
2000s, when targeted drugs, like cetuximab and bevacizumab, were developed. The use of targeteddrugs in clinical practice has significantly increased patients’ overall survival. To date, the emergenceof several types of targeted drugs has opened new possibilities and revealed new prospects for mCRC
treatment. Therapeutic strategies are continually being updated to select the most suitable targeteddrugs based on the results of clinical trials that are currently underway. This review discusses the
up-to date molecular evidence of targeted therapy for mCRC and summarizes the Food and Drug
Administration-approved targeted drugs including the results of clinical trials. We also explain theirmechanisms of action and how these affect the choice of a suitable targeted therapy.

Kawada, M. Current Targeted
Therapy for Metastatic Colorectal

Keywords: colorectal cancer; targeted therapy; clinical trial

Cancer. Int. J. Mol. Sci. 2023, 24, 1702.
https://doi.org/10.3390/
ijms24021702
Academic Editors: Philippe

1. Introduction

Pourquier and Zhuoxun Wu

Colorectal cancer (CRC) arises from the epithelial cells lining the colon or rectumof the gastrointestinal tract and is the third most common cancer type among men andwomen in the United States [1]. CRC is also the third most commonly diagnosed cancer andwas the second leading cause of cancer deaths in 2020 worldwide [2]. Surgery or surgery
plus radiotherapy and chemotherapy in the adjuvant setting has improved the survivalof patients with CRC, and the 5-year survival rate for CRC is 65%. However, because thisfalls to 15% for metastatic CRC (mCRC), the development of new therapeutic approaches
to mCRC are critical [1,3].
Although complete surgical resection of the tumor and its metastatic sites improves
overall survival (OS) in patients with CRC, approximately 25% of CRCs are diagnosed at anadvanced stage with metastases in distant organs, which is difficult to manage surgically [4].
Unresectable advanced or recurrent CRC is treated with chemotherapy along with targeted
therapy and/or radiotherapy to reduce the tumor size and prolong patient survival [5].
Regarding chemotherapy and targeted therapy, there are several first-line therapeutic
options, and understanding the gene mutation status in CRC and resistance mechanisms is

Received: 2 November 2022
Revised: 7 January 2023
Accepted: 13 January 2023
Published: 15 January 2023

Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Int. J. Mol. Sci. 2023, 24, 1702. https://doi.org/10.3390/ijms24021702

https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023, 24, 1702

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crucial to choose the best therapeutic option [6]. Notably, on rare occasions, the treatment
may facilitate tumor downstaging, thereby improving the opportunity for resection.
Cytotoxic chemotherapy remains the standard treatment strategy for mCRC. Fluoropyrimidines play an important role as the backbone of combination regimens. Chemotherapy,
such as FOLFOX (fluorouracil, leucovorin, and oxaliplatin), FOLFIRI (fluorouracil, leucovorin, and irinotecan), or FOLFOXIRI (fluorouracil, leucovorin, oxaliplatin, and irinotecan),combined with or without targeted drugs (anti-epidermal growth factor receptor [EGFR]
antibody or anti-vascular endothelial growth factor [VEGF] antibody) is considered thefirst-line treatment for mCRC [7]. When chemotherapy is used, the doctor should give thepatient as much information as possible about side effects because their severity depends on
various factors, such as the type of cytotoxic drugs used and the duration of treatment [8,9].
Therefore, the active management of side effects is crucial so that the patient can continue
chemotherapeutic treatment.
Several targeted drugs have been developed and studied. These drugs target themolecules involved in tumorigenesis and their related signaling pathways in cancer cells
that make them different from normal cells [10]. Additionally, the tumor microenvironment,
including blood vessels in the tissue surrounding the tumor and immune cells, are also
affected by these targeted drugs to impede tumor growth and improve antitumor immune
surveillance and attack [11]. The major types of targeted drugs are monoclonal antibodies
and small molecule inhibitors. Such drugs are advantageous because, unlike chemotherapy,
they can be chosen based on the molecular characteristics of tumor types [12]. However,a large number of patients experience disease progression even after receiving standard
regimens. In the future, personalized medicine, in which drugs and drug combinations
are optimized based on each patient’s available data including their genetic and epigenetic
features and alterations, will improve the efficacy of treatments, reduce side effects, andbenefit cancer patients [13,14].
This review summarizes the up-to-date evidence of clinical successes using targeted
therapies to treat patients with mCRC. The molecular mechanisms of action and how these
affect the choice of a suitable targeted therapy are also discussed.
2. mCRC Treatment Strategies
In the 1990s, fluorouracil-based chemotherapy improved the OS of patients with
mCRC to 14 months. Later, the additional combination of leucovorin and oxaliplatin(FOLFOX) prolonged the OS to 19.5 months [15,16]. In 2004, the first Food and Drug
Administration (FDA)-approved targeted drug was the anti-EGFR antibody cetuximab [17].
Since then, many targeted drugs for mCRC have been approved by FDA (Table 1).
Table 1. FDA-approved targeted drugs for mCRC.
Year Approved by
the FDA
2004
2006

Drugs

Targets

Drug Details

Ref

Cetuximab

EGFR

Chimeric mouse/human mAb (IgG1 )[18]

Bevacizumab

VEGF-A

Humanized mAb (IgG1 )[18]

Panitumumab

EGFR

Fully human mAb (IgG1 )[19]

Aflibercept

VEGF-A, VEGF-B, PlGF

Fusion protein which consists of thebinding portions of VEGF from
VEGF-1 and 2 fused to the Fc portion
ofimmunoglobulin G1 (IgG1 )[20]

Regorafenib

VEGFR, FGFR, KIT,PDGFR, BRAF

Small molecule inhibitor ofmembrane-bound and intracellular
receptor tyrosine kinases

[21]

2012

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Table 1. Cont.
Year Approved by
the FDA

Drugs

Targets

Drug Details

Ref

2015

Ramucirumab

VEGFR-2

Fully human mAb (IgG1 )[22]

Pembrolizumab

PD-1

Humanized mAb (IgG4 )[23]

Nivolumab

PD-1

Fully human mAb (IgG4 )[24]

Ipilimumab

CTLA-4

Fully human mAb (IgG1 )[25]

Larotrectinib

TRK

Small molecule of tyrosine
3 of 20
kinase inhibitor

[26]

2017

2018

2019
2020

Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW

2015

Entrectinib
Ramucirumab

TRK, ALK, ROS1

Small molecule of tyrosine[27]

VEGFR-2
Fully human mAbkinase(IgG1) inhibitor[22]
Pembrolizumab
PD-1
Humanized mAb (IgG4)[23]
2017Encorafenib
BRAF
Small
molecule[28]
Nivolumab
PD-1 (V600E-mutant) Fully humanmAb(IgG4) kinase inhibitor[24]
Ipilimumab
CTLA-4
Fully
human
mAb
(IgG
1)
[25]
Abbreviations: EGFR: epidermal growth factor receptor; VEGF: vascular endothelial growth factor; PlGF: placenta
2018
TRK endotheliaSmall
molecule
tyrosine kinase[26]
growth Larotrectinib
factor; VEGFR: vascular
growthfactorofreceptor;
FGFR:inhibitor
fibroblast growth
factor receptor; KIT:
2019
Entrectinib
TRK,
ALK,receptor;
ROS1
Small moleculeof tyrosine growthkinase inhibitor
mast/stem
cell growth
factorPDGFR:
platelet-derived
factor receptor;[27]BRAF: v-Raf murine(V600EsarcomaEncorafenib
viral oncogeneBRAF
homolog
B1; PD-1: programmed
cell death
CTLA-4: cytotoxic T-lymphocyte-associated2020
Small moleculekinase1;inhibitor[28]
mutant)protein-4; TRK: tropomyosin
receptor kinase; ALK: anaplastic lymphoma kinase; ROS1: c-ros oncogene 1; mAb:
Abbreviations: EGFR: epidermal growth factor receptor; VEGF: vascular endothelial growth factor;
monoclonal antibody.

PlGF: placenta growth factor; VEGFR: vascular endothelia growth factor receptor; FGFR: fibroblast
growth factor receptor; KIT: mast/stem cell growth factor receptor; PDGFR: platelet-derived growth
factor receptor;
v-Raf murine
sarcoma involves
viral oncogenemediation
homolog B1; PD-1:
programmedcell
The progression
andBRAF:
spreadof mCRCwithreceptors
in several
death 1 ; CTLA-4: cytotoxic T-lymphocyte-associated protein-4; TRK: tropomyosin receptor kinase;signaling pathways.
These
includeEGFRs,
fibroblast
growthfactorreceptors(FGFRs),
ALK: anaplastic lymphoma kinase; ROS1: c-ros oncogene 1; mAb: monoclonal antibody.

vascular endothelia growth factor receptors (VEGFRs), and tropomyosin receptor kiThe progression and spread of mCRC involves mediation with receptors in several
nases (TRKs) [29,30].
Furthermore,
tumor
cells
express
signaling pathways.
These include
EGFRs,
fibroblast
growthB7-1
factor(CD80)/B7-2
receptors (FGFRs),(CD80) andvascularendothelia
factor receptors(VEGFRs),
andtropomyosin
receptor kinases
programmed cell
death
ligandgrowth
1 (PD-L1),
whichbind tocytotoxic
T-lymphocyte-associated(TRKs) [29,30]. Furthermore, tumor cells express B7-1 (CD80)/B7-2 (CD80) and proprotein-4 (CTLA-4)
and programmed cell death 1 (PD-1), respectively, on T cells to escapegrammed cell death ligand 1 (PD-L1), which bind to cytotoxic T-lymphocyte-associatedimmune surveillance[30]. Therefore,
these
their related
pathways
must beprotein-4 (CTLA-4)
and programmed
cellmolecules
death 1 (PD-1),and
respectively,
on T cells toescapeimmune surveillance[30]. Therefore,
inhibited by targeteddrugs (Figure1). these molecules and their related pathways must be
inhibited by targeted drugs (Figure 1).

Figure 1. Comprehensive overview of FDA-approved targeted drugs for mCRC. Abbreviations:Figure 1. Comprehensive
overview
ofreceptor;
FDA-approved
drugs
for
mCRC.
Abbreviations:
EGFR: epidermal
growth factor
FGFR: fibroblasttargeted
growth factor
receptor;
VEGFR:
vascularfactorreceptor; TRK:
tropomyosin
receptorkinase; factor
VEGF: vascularendothelial
EGFR: epidermalendothelia
growthgrowth
factorreceptor;
FGFR:
fibroblast
growthreceptor;
VEGFR: vascular
growth factor; PlGF: placenta growth factor; PD-1: programmed cell death 1; PD-L1: programmed
endothelia growth
receptor;
TRK:
tropomyosin
receptorprotein-4;
kinase;B7:VEGF:
vascularcell factor
death ligand
1; CTLA-4:
cytotoxic
T-lymphocyte-associatedCD80/CD86;
MEK: endothelial
mitogen-activated protein kinase; ERK: extracellular signal-regulated kinase; PI3K:
growth factor; PlGF:
placenta growth factor; PD-1: programmed cell death 1; PD-L1: programmed
cell death ligand 1; CTLA-4: cytotoxic T-lymphocyte-associated protein-4; B7: CD80/CD86; MEK:
mitogen-activated protein kinase; ERK: extracellular signal-regulated kinase; PI3K: phosphoinositide
3-kinase; mTOR: mechanistic target of rapamycin; PLC-γ: phospholipase C-γ; PKC: protein kinase C;
NF-kB: nuclear factor-kappa B; JAK: Janus kinase; STAT: transducer and activator of transcription.

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3. EGFR-Targeting Strategy
3.1. Molecular Mechanism of EGFR Signaling
EGFR is a member of the ErbB family of receptors, a subfamily of four receptor tyrosine
kinases, including EGFR (ErbB-1), HER2 (ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4). It
consists of extracellular, transmembrane, and intracellular domains and regulates cell proliferation, survival, differentiation, and migration [31]. The binding of ligands to the extracellular domain of EGFR promotes receptor dimerization, activating downstream signaling
pathways, such as RAS/Raf/mitogen-activated protein kinase (MEK)/extracellular signalregulated kinase (ERK), phosphoinositide 3-kinase (PI3K)/Akt, Janus kinase (JAK)/signaltransducer and activator of transcription (STAT), and phospholipase C (PLC)-γ/protein
kinase C (PKC), leading to the activation of gene transcription and playing key roles incancer initiation and progression [32–35]. Several tumors, including mCRCs, overexpress
EGFR, and the aberrant EGFR signaling is associated with poor prognosis. Thus, thisreceptor is a promising target for mCRC treatment [36].
3.2. Cetuximab and Panitumumab
Cetuximab and panitumumab are FDA-approved agents targeting EGFR (Table 1 andFigure 1). They are both distinct monoclonal antibodies and are used in monotherapy or in
combination therapy with chemotherapy to treat patients with RAS wild-type mCRC [37].
Multiple reports have demonstrated that responses to either cetuximab or panitumumaboccur exclusively in patients with mCRC without mutations in KRAS and NRAS codons 12
and 13 of exon 2, codons 59 and 61 of exon 3, and codons 117 and 146 of exon 4 [38].
Cetuximab is a chimeric mouse-human monoclonal antibody of the IgG1 subclass.
It had the highest capacity to stimulate antibody-dependent cell-mediated cytotoxicity(ADCC) compared with other isotypes (such as IgG2 , IgG3 , and IgG4 ) [39,40]. It is thoughtthat ADCC is mainly mediated by natural killer cells or macrophages, and it is one of
the important modes of action of therapeutic antibodies [41]. Cetuximab showed great
potential in an initial phase II clinical trial [42]. This was also confirmed by a randomized
phase II trial (BOND trial) of cetuximab plus irinotecan or single irinotecan, which reported
an OS of 22.9 months (218 subjects) vs. 10.8 months (111 subjects), respectively, in irinotecanrefractory patients [43] (Table 2).
Table 2. Selected clinical trials for EGFR (± BRAF)-targeting drugs.
Targeted Drugs

Cetuximab

Panitumumab

Cetuximab +Encorafenib

Study

BOND

PRIME

BEACON

Phase

Study Regimen

II

Cetuximab +Irinotecan/Irinotecan(Refractory to
Irinotecan)(Third-line)

III

Panitumumab +FOLFOX4/FOLFOX4(First-line)

III

Cetuximab +Encorafenib/Cetuximab +Chemotherapy 1(Second- or
later-line)

NumberResults

Ref

218/111OS:
22.9/10.8 months(p = 0.007)
Disease control:
55.5%/32.4%(p < 0.001)

Cunningham et al. [43]325/331OS:
23.8/19.4 months(p = 0.03)
PFS:
10.0/8.6 months(p = 0.01)

Douillard et al. [44]220/221Median OS:
8.4/5.4 months(p < 0.001)
Confirmed RR:
20%/2%(p < 0.001)

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Table 2. Cont.
Targeted Drugs

Cetuximab +Encorafenib +Binimetinib

Study

BEACON

Phase

Study Regimen

III

Cetuximab +Encorafenib +Binimetinib/Cetuximab +Chemotherapy 1(Second- or
later-line)

NumberResultsRef

224/221Median OS:
9.0/5.4 months(p < 0.001)
Confirmed RR:
26%/2%(p < 0.001)

Kopetz et al. [45]1 The investigators’ choice of either cetuximab and irinotecan or cetuximab and FOLFIRI (folinic acid, fluorouracil,

and irinotecan. Abbreviations: OS: overall survival; PFS: progression-free survival; RR: response rate.

Panitumumab is a fully human monoclonal IgG2 antibody. Whereas cetuximab, achimeric mouse-human monoclonal antibody, might induce immunogenic reactions, thereis less fear of this happening with panitumumab [46]. In fact, panitumumab was reported
to show a lower risk of hypersensitivity reactions than cetuximab [47]. On the other hand,unlike cetuximab, panitumumab does not induce ADCC [48]. In a randomized phase
III trial (PRIME trial), panitumumab plus FOLFOX4 showed improved progression-freesurvival (PFS) and OS of patients with mCRC compared with FOLFOX4 alone [44] (Table 2).
Importantly, cetuximab and panitumumab recognize and bind to domain III of EGFR
and are effective in patients with wild-type RAS mCRC; both agents showed similar OS
in a phase III trial (ASPECCT trial) [49,50]. Panitumumab shows effectiveness following
cetuximab failure, and cetuximab is effective following panitumumab failure, indicatingthat the mechanisms of action of these two agents differ [51,52].
Although cetuximab and panitumumab have been used to treat mCRC, their use islimited to patients with wild-type RAS because patients with RAS mutations do not benefit
from anti-EGFR treatment [53–65]. Notably, although approximately 50% of patients with
CRC harbor RAS mutations (KRAS: 36% and NRAS: 3%) [66], not all patients with KRAS
mutations are resistant to EGFR-targeted therapy [67]. There are conflicting reports withrespect to the KRAS codon G13D mutation. Several retrospective studies demonstrated
that cetuximab confers clinical benefits to patients with KRAS codon G13D-mutated mCRC
compared with patients harboring other KRAS mutations [68,69]. A cell line from a tumor
with a KRAS codon G12V mutation was unresponsive to cetuximab and panitumumab,
whereas cell lines from a tumor with a KRAS codon G13D mutation showed an intermediate
responsive to cetuximab and panitumumab in comparison with resistant KRAS codon
mutation G12V and wild-type cells [70]. De Roock et al. investigated the association of
response and survival between patients with KRAS codon G13D and other mutations (data
set of 579 patients with chemotherapy-refractory CRC treated with cetuximab). They foundthat cetuximab treatment was associated with longer PFS and OS [71]. Although this finding
was obtained from a retrospective study, in a randomized phase II study, Nakamura et al.
showed that cetuximab-based treatment benefited patients with chemotherapy-resistant,refractory KRAS codon G13D-mutated mCRC [72]. Because of the limitations of the low
number of patients with KRAS mutations in the datasets, further clinical studies withlarger sample sizes are necessary to evaluate the differences in the efficacy of the EGFRtargeting strategy for the patients with the KRAS codon G13D mutation and those with
KRAS mutations other than codon G13D.
It is important to note that tumor-sidedness has been identified as an important prognostic factor found in several clinical studies [73,74]. It has been reported that patientswith wild-type KRAS/RAS having right-sided tumors are associated with worse prognosis to anti-EGFR treatment than left-sided tumors regardless of the first-line treatment
given [74,75]. In addition, retrospective analyses have revealed that wild-type RAS withright-sided mCRC also leads to poor prognosis in second and later line anti-EGFR treatments than left-sided tumors [76]. Since the left and right colon have different embryologic
origins, the left colon is from the foregut and right from midgut, this may explain the
difference in treatment effects. Based on the results and randomized clinical trial results

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above, patients with right-sided mCRC with wild-type KRAS/NRAS/BRAF mutations
should use anti-EGFR containing chemotherapy regimens [77,78].
Rechallenge with anti-EGFR treatment in patients who have responded to first-line
regimens containing an EGFR targeting drug is emerging as a valid therapeutic strategy inpatients with mCRC. In a phase II single-arm clinical trial (CRICKET trial), Cremolini et al.
demonstrated that cetuximab rechallenge strategy is beneficial in patients with RAS and
BRAF wild-type mCRC with acquired resistance to first-line cetuximab-based treatment(median PFS, 4.0 vs. 1.9 months, p < 0.001) [79]. In this trial, liquid biopsies are used toassess the circulating tumor DNA (ctDNA), and it is found that only patients with no RAS
and BRAF mutations detected in their ctDNA may have a clinical benefit by the rechallengewith anti-EGFR treatment. In a single arm, open-label phase II study (CHRONOS trial),
compared with standard third-line treatments, liquid biopsy-driven anti-EGFR rechallengewith panitumumab maximizes the therapeutic effects and avoids adverse effects [80].
4. VEGF/VEGFR Targeting Strategy
Angiogenesis is the process whereby new vessels are formed or reformed from existing vessels, and tumor angiogenesis plays an important role in tumor growth. The
VEGF/VEGFR signaling pathway is recognized as one of the most predominant factors
contributing to tumor angiogenesis, which participates in the multiple processes of tumor
progression by activating host vascular endothelial cells [81].
Although, a VEGF/VEGFR-targeted strategy has been used in the patients with CRC
with or without RAS mutations, patients with left-sided mCRC and right-sided mCRC
with KRAS/NRAS/BRAF mutations should be considered for a VEGF/VEGFR-targeted
drug containing chemotherapy regimens.
4.1. Molecular Mechanism of VEGF/VEGFR Signaling
VEGF family proteins and VEGFRs are key factors of tumor growth and metastasis that
regulate normal and pathological tumor angiogenesis, leading to the activation of several
signaling pathways [81]. The VEGF family consists of five members (VEGF-A, B, C, D, andplacenta growth factor [PlGF]) that bind to endothelial cells through VEGFRs, including
VEGFR-1, VEGFR-2, and VEGFR-3 [82]. While VEGF-A, VEGF-B, and PlGF mainly induce
angiogenesis, VEGF-C and VEGF-D tend to regulate lymphangiogenesis [83]. VEGF-A,
VEGF-B, and PlGF bind to VEGFR-1; VEGF-A, VEGF-C, and VEGF-D bind to VEGFR-2;
and VEGF-C and VEGF-D bind to VEGFR-3; respectively, leading to various biologicalresponses [84]. VEGFs cause VEGFR dimerization, which activates intrinsic tyrosine kinase,leading to the activation of signaling pathways, such as RAS/Raf/MEK/ERK, PI3K/Akt,and PLC-γ/PKC, to enhance tumor angiogenesis and proliferation. Among them, VEGFR-1
and VEGFR-2, which are common receptors for VEGF-A, are considered promising targets
against cancer in clinical settings [85,86].
4.2. Bevacizumab
Bevacizumab is a humanized anti-VEGF-A monoclonal IgG1 antibody that inhibits
VEGF-A binding to VEGFR-1 and VEGFR-2, and it is approved by the FDA to treat
mCRC [87] (Table 1 and Figure 1). Hurwitz et al. demonstrated that relative to placebo
plus IFL (irinotecan, fluorouracil, and leucovorin), the addition of bevacizumab to IFL
when treating patients with mCRC significantly improved the 1-year survival rate (74.3% in
402 subjects vs. 63.4% in 411 subjects), OS (20.3 vs. 15.6 months), PFS (10.6 vs. 6.2 months),
and response rate (RR) (44.8% vs. 34.8%) [88] (Table 3). In a randomized phase III trial(AVEX study), the addition of bevacizumab to capecitabine showed a tolerable safety profileand efficient administration and significantly improved OS (20.7 months in 140 subjects vs.
16.8 months in 140 subjects) and PFS (9.1 vs. 5.1 months) relative to single capecitabine inelderly patients (aged > 70 years) [89] (Table 3). Furthermore, in a randomized phase III trial(TRIBE trial), Cremolini et al. showed that a combination regimen of bevacizumab with

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the FOLFOXIRI showed better efficacy than that with FOLFIRI (OS: 31.0 vs. 25.8 months,
p = 0.125; PFS: 12.1 vs. 9.7 months, p = 0.006; RR: 65% vs. 53%, p = 0.006; respectively) [90].
Table 3. Selected clinical trials for VEGF/VEGFR-targeting drugs.
Targeted Drugs

Bevacizumab

Bevacizumab

Study

Phase

Clinical study

AVEX

Aflibercept

VELOUR(NCT00561470)

Regorafenib

CORRECT(NCT01103323)

Regorafenib

CONCUR(NCT01103323)

Ramucirumab

RAISE(NCT01183780)

Study Regimen

III

Bevacizumab +IFL/Placebo +IFL(First-line)

III

Bevacizumab +Capecitabine/
Capecitabine(First-line)

III

Aflibercept +FOLFILI/Placebo+ FOLFILI(Second-line)

III

Regorafenib/Placebo(Third- or
later-line)

III

Regorafenib/Placebo(Third- or
later-line)

III

Ramucirumab +FOLFIRI/Placebo+ FOLFIRI(Second-line)

NumberResults

Ref

402/411OS:
20.3/15.6 months(p < 0.001)
PFS:
10.6/6.2 months(p < 0.001)
RR: 44.8%/34.8%(p = 0.004)
1-year survival rate:
74.3%/63.4%(p < 0.001)

Hurwitz et al.
[88]

140/140OS:
20.7/16.8 months(p = 0.18)
PFS: 9.1/5.1 months(p < 0.001)

Cunningham et al.
[89]

612/614OS:
13.5/12.06 months(p = 0.0032)
PFS:
6.9/4.67 months(p < 0.0001)
RR: 19.8%/11.1%(p = 0.0001)

Van Cutsem et al.
[91]

505/255

OS: 6.4/5.0 months(p = 0.0052)
PFS: 1.9/1.7 months(p < 0.0001)

Grothey et al.
[92]

138/68

OS: 8.8/6.3 months(p = 0.00016)
PFS: 3.2/1.7 months(p < 0.0001)

Li et al. [93]536/536OS:
13.3/11.7 months(p = 0.0219)
PFS: 5.7/4.5 months(p < 0.0005)

Tabernero et al.
[94]

Abbreviations: OS: overall survival; PFS: progression-free survival; RR: response rate.

Although some clinical trials using bevacizumab with chemotherapy showed partial
improvement in OS or PFS, bevacizumab in combination with chemotherapy is similarly
effective in patients with KRAS wild-type and KRAS mutant mCRC [95]. In addition,
Yoshimatsu et al. showed that there was no difference in therapeutic effect of bevacizumab
for mCRC patient with RAS wild-type and RAS mutant [96]. Although both the EGFR andVEGF/VEGFR signaling pathways have been identified as possible therapeutic targets totreat patients with KRAS wild-type mCRC, data from several clinical trials (FIRE-3 trial
and PEAK trial) shows that anti-EGFR treatment (cetuximab or panitumumab) appears
superior to anti-VGFR treatment (bevacizumab) [77,78].
Importantly, Grothey et al. performed two large observational cohort studies andshowed that after failure of bevacizumab combination chemotherapy for first-line treatment,
continued bevacizumab beyond initial progression (BBP) improves OS and post progression
survival [97,98]. In addition, during a randomized phase III trial (ML18147), relative tochemotherapy alone, second-line changing chemotherapy with bevacizumab at the same
dose as used in the first-line treatment beyond disease progression improved median OS(11.2 months in 409 subjects vs. 9.8 months in 411 subjects) and PFS (6.9 vs. 4.67 months) [99].
From several other clinical studies including ARIES [100], RAISE [94], and VELOUR [101],

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BBP might have clinical benefits in patients who have experienced disease progression
during a first-line bevacizumab containing chemotherapy.
4.3. Aflibercept
Aflibercept (also known as ziv-aflibercept) is a soluble molecule composed of thecritical ligand-binding domains of human VEGFR-1 and VEGFR-2 fused with the Fc
fragment of human IgG1 . It functions as a decoy receptor by binding to VEGF-A, VEGFB, and PlGF [102,103] (Table 1 and Figure 1). Aflibercept binds to VEGF-A with higher
affinity and a faster association rate than bevacizumab [103]. In a randomized phase III
trial (VELOUR trial), relative to placebo plus FOXFIRI, aflibercept plus FOXFIRI regimen
improved OS (13.5 months in 612 subjects vs. 12.06 months in 614 subjects), PFS (6.9
vs. 4.67 months), and RR (19.8% vs. 11.1%) in patients with mCRC who progressed after
receiving an oxaliplatin-based regimen [91] (Table 3). From these results, the FDA-approved
aflibercept for the treatment of mCRC when given in combination with the FOLFIRI in
2012. However, in a randomized phase II trial (AFFIRM study), adding aflibercept tofirst-line modified FOLFOX6 (mFOLFOX6) did not show significant efficacy (aflibercept
plus mFOLFOX6 in 119 subjects vs. mFOLFOX6 in 116 subjects, PFS: 8.48 vs. 8.77 months,
RR: 49.1% vs. 45.9%). Use as a second-line treatment after progression following first-line
treatment, aflibercept in combination with FOLFIRI showed efficacy following bevacizumab
plus FOLFOXIRI for unresectable or mCRC in single arm phase II trials [104]. Therefore,
the use of aflibercept-based regimens is recommended in second-line settings.
4.4. Ramucirumab
Ramucirumab is a fully human anti-VEGF-A monoclonal IgG1 antibody that inhibits
VEGFR-2 and its downstream angiogenesis pathways. It was approved by the FDA for
second-line use in combination with FOLFIRI for patients with mCRC who progressed
during or after treatment with bevacizumab, oxaliplatin, and fluoropyrimidine [105]. Ina second-line randomized phase III study (RAISE), relative to the placebo, ramucirumabin combination with FOLFIRI significantly improved OS (13.3 months in 536 subjects) vs.
(11.7 months in 536 subjects) and PFS (5.7 vs. 4.5 months) with FOLFIRI [94] (Table 3). This
was also confirmed by a retrospective study of ramucirumab plus FOLFIRI as a second- or
later line therapy, which reported a positive impact on survival outcomes, with the PFS
on second-line ramucirumab of 5.4 months (26 subjects) being equivalent to that observedin the RAISE trial [106]. Additionally, another retrospective study of ramucirumab plus
FOLFIRI as second-line therapy reported an OS of 17.0 months (74 subjects) and PFS of
6.2 months (74 subjects), which was also equivalent to the RAISE trial [107].
4.5. Regorafenib
Regorafenib is a multikinase inhibitor that inhibits multiple intracellular and membranebound receptor tyrosine kinases, including VEGFR and FGFR involved in the regulation oftumor angiogenesis. It was approved by the FDA to treat previously treated patients with
mCRC [108] (Table 1 and Figure 1). In a clinical phase III trial (CORRECT trial), Grothey
et al. demonstrated that compared with the placebo, regorafenib significantly improved theOS (6.4 months in 505 subjects vs. 5.0 months in 255 subjects) and PFS (1.9 vs. 1.7 months) intreatment-refractory mCRC [92] (Table 3). This was also confirmed by a randomized phase
III trial (CONCUR trial) of regorafenib plus best supportive care (BSC) or placebo plus BSC,
which reported an OS of 8.8 months (138 subjects) vs. 6.3 months (68 subjects) and PFS of
3.2 vs. 1.7 months [93] (Table 3). Tyrosine kinase inhibitors (TKIs) have become one of thestandard treatment regimens for patients with non-small-cell lung cancers [109]. Several
TKIs have been tested for the patients with mCRC in clinical settings over recent years.
4.6. Fruquintinib
Fruquintinib is a potent and selective small molecule tyrosine kinase inhibitor of
VEGFR 1, 2, and 3 [110]. It was approved in China for use in the treatment of mCRC.

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The FDA granted a fast track designation for the development of fruquintinib for thetreatment of patients with mCRC who have received prior fluoropyrimidine-, oxaliplatin-,and irinotecan-based chemotherapy; EGFR-targeted therapy; and VEGF-targeted therapy.
In a randomized, multicenter, phase III FRESCO trial conducted in China (NCT02314819),
fruquintinib treatment in patients with mCRC who progressed after standard secondline therapy showed significant improvement in the PFS and OS [111]. Furthermore,in a randomized phase III FRESCO-2 trial (NCT04322539), with a total of 687 patientswith refractory mCRC for more than third-line treatment, fruquintinib treatment showedsignificant PFS and OS [112]. These results indicate the clinical benefit of Fruquintinib forthe patients with mCRC beyond third-line therapy.
5. Immune Checkpoint Targeting Immunotherapies
Immunotherapy is a novel treatment option against several types of tumors. Tumor
immunotherapy induces an immune cell-mediated immune response through the neoantigen expressed on a broad range of tumors [113]. The success of tumor immunotherapy inachieving long-lasting antitumor responses has demonstrated that immune cells, mainly T
cells, could be utilized to eliminate tumor cells [114].
Microsatellite instability (MSI) is an indicator of defective DNA mismatch repair(dMMR) and an MSI/dMMR status is observed in approximately 5% of mCRC cases [115].
Because MMR pathways are responsible for correcting DNA replication errors, MSI-hightumors carry various somatic mutations, leading to a high neoantigen exposure that favors
the initiation of an antitumor immune response [116,117]. Therefore, MSI-high tumors
respond well to immunotherapy.
5.1. Molecular Mechanism of Immune Checkpoints
Programmed cell death-1 (PD-1) and CTLA-4 are expressed on the surface of activatedimmune cells, including T cells, and are key immune checkpoint molecules that inactivate immune cells through distinct mechanisms [118]. PD-1 inhibits T cell response via
interaction with its two ligands, PD-L1 and PD-L2, to mediate an inhibitory signal in T
cells, which suppresses cellular and humoral immune responses [119]. Blockage of PD-1 orPD-L1 can inhibit the checkpoint and induce T cell activation to drive immunity [120,121].
CTLA-4 is expressed on activated T cells and regulatory T cells (Tregs). It inhibits
T cell response by interacting with B7-1 and B7-2 ligands to provide an inhibitory signal
in T cells [122]. Inhibition of CTLA-4 binding to these ligands results in the reactivationand proliferation of T cells and decreases immunosuppressive Tregs, leading to increased
activation of the immune system in the tumor microenvironment [123].
5.2. Pembrolizumab, Nivolumab, and Ipilimumab
Pembrolizumab is a humanized IgG4 anti-PD-1 monoclonal antibody and nivolumab
is a human IgG4 anti-PD-1 monoclonal antibody. They have been approved by the FDA
for patients with multiple tumors, including MSI-high/dMMR mCRC [124] (Table 1 andFigure 1). In a phase II study (NCT01876511), pembrolizumab treatment in patients with
MSI-high/dMMR mCRC in second-line settings showed an objective RR (ORR) of 52%(40 subjects), disease control rate of 82%, 2-year OS of 72%, and 2-year PFS of 59% [125](Table 4). Furthermore, in an open-label phase III trial (KEYNOTE-177), pembrolizumab asfirst-line treatment for patients with MSI-high/dMMR mCRC was superior to chemotherapy with respect to PFS (16.5 months in 153 subjects vs. 8.2 months in 154 subjects) andORR (43.8% vs. 33.1%). Importantly, treatment-related adverse effects of grade 3 or higher
occurred in 22% of the patients in the pembrolizumab-treated group compared with 66%
in the chemotherapy-treated group [126] (Table 4). In addition to pembrolizumab, in an
open-label phase II trial (CheckMate 142), second-line treatment with nivolumab in patientswith MSI-high/dMMR mCRC showed an ORR of 31% (74 subjects) and disease control
for 12 weeks or longer of 69% [127] (Table 4). From these results, pembrolizumab andInt. J. Mol. Sci. 2023, 24, 1702

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nivolumab are thought to be effective and show a manageable safety profile in patientswith MSI-high/dMMR mCRC.
Table 4. Selected clinical trials for immune checkpoint-targeting drugs.
Targeted Drugs

Pembrolizumab

Pembrolizumab

Nivolumab

Nivolumab+Ipilimumab

Study

Clinical study(NCT01876511)

KEYNOTE-177(NCT02563002)

CheckMate-142(NCT02060188)

CheckMate-142(NCT02060188)

Phase

Study Regimen

II

Pembrolizumab(Second-line)(Patients withMSIhigh/dMMR
mCRC)

NumberResults

Ref

40

ORR: 52%
Disease control
rate: 82%2-year OS: 72%
2-year PFS: 59%

Le et al.
[125]

II

Pembrolizumab/
Chemotherapy(First-line)(Patients withMSIhigh/dMMR
mCRC)

153/154

PFS: 16.5/8.2
monthsORR:
43.8%/33.1%

Andre et al.
[126]

II

Nivolumab(Second-line)(Patients withMSIhigh/dMMR
mCRC)

74

ORR: 31%
Disease control
for 12 weeks orlonger: 69%

Overman et al.
[127]

II

Nivolumab + Ipilimumab/Nivolumab(Second-line)(Patients withMSIhigh/dMMR
mCRC)

119/741-year OS:
85%/73%
ORR: 55%/31%
Disease control
for 12 weeks orlonger: 80%/69%

Overman et al.
[128]

Abbreviations: ORR: objective response rate; OS: overall survival; PFS: progression-free survival.

Ipilimumab is a fully human IgG1 anti-CTLA-4 monoclonal antibody. It has been
approved by the FDA for combination therapy with nivolumab in patients with MSIhigh/dMMR mCRC after progression following chemotherapy [25] (Table 1 and Figure 1).
In an open-label phase II trial (CheckMate-142), relative to nivolumab, nivolumab in
combination with ipilimumab in patients with MSI-high/dMMR mCRC improved 1-year
OS (85% in 119 subjects vs. 73% in 74 subjects), ORR (55% vs. 31%), and disease control
for 12 weeks or longer (80% vs. 69%) [128] (Table 4). These results indicated that the
combination of nivolumab and ipilimumab showed a favorable impact on the quality oflife for patients with MSI-high/dMMR mCRC.
6. NTRK Signaling Pathway and Targeting Strategy
The neurotrophic tropomyosin receptor kinases (NTRK) family consists of three members (NTRK1, NTRK2, and NTRK3) that code for TRKA, TRKB, and TRKC, respectively,
and they are mainly expressed in neural and neuronal tissues [129]. They perform biological functions by homodimerization, which activates their downstream pathways, including
RAS/Raf/MEK/ERK, PI3K/Akt, and PLC-γ/PKC, leading to the activation of gene transcription, cell survival, and progression [130,131]. Multiple NTRK fusions are one of themost common oncogenic events, leading to constitutive activation of their downstream
pathways [132]. Besides NTRK, anaplastic lymphoma kinase (ALK) and c-ros oncogene
1 (ROS1) fusions also occur in CRC. ALK and ROS1 also encode tyrosine kinases that are
constitutively activated by fusion, leading to the activation downstream signaling pathways
for tumor cell growth and progression [133,134]. Their gene rearrangements are thought
to be mutually exclusive in lung cancers despite ALK and ROS1 tyrosine kinase domains
sharing high homology [135]. Although these fusions occur in less than 2.5% of CRC cases,
they are considered oncogenic drivers and a potential target for mCRC treatment [136,137].

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Larotrectinib and Entrectinib
Larotrectinib and entrectinib are first-generation TRK inhibitors. They were approvedby the FDA for patients with solid tumors that harbor NTRK gene fusions [138] (Table 1).
Larotrectinib is a selective inhibitor of TRK that was tested in three phase I/II clinical trialsof 153 cancer patients carrying NTRK fusions (age: 48–67 years) and showed an impressive
ORR of 79% and good tolerability [139] (Figure 1).
Entrectinib is a potent TRK, ROS1, and ALK kinase inhibitor that was tested in threephase I/II clinical trials of 54 cancer patients carrying NTRK fusions (age: 1 month–84 years)
and showed an impressive ORR of 57% and good tolerability [137] (Figure 1).
7. BRAF Signaling Pathway and Targeting Strategy
BRAF is a downstream effector of RAS in the RAS/Raf/MEK/ERK signaling pathway
and is a well-known oncogenic driver [140]. Mutations in BRAF V600E are present inapproximately 10% of mCRCs and associated with chemotherapy resistance and worse
prognosis [141,142]. Interestingly, non-V600 BRAF mutation was independently and significantly associated with improved OS [142]. Given that monotherapy with a BRAF-targeting
tyrosine kinase inhibitor for patients with mCRC has failed, some combination regimens
have been tested in multiple clinical trials [143].
Encorafenib
Encorafenib is a kinase inhibitor that targets BRAF V600E as well as wild-type BRAF
and shows more prolonged pharmacodynamic activity than other BRAF inhibitors [144](Table 1 and Figure 1). In an open-label phase III trial (BEACON), compared with the control(cetuximab plus chemotherapy), encorafenib in combination with cetuximab showed asignificantly improved median OS (8.4 months in 220 subjects vs. 5.4 months in 221 subjects)
and confirmed RR (20% vs. 2%) [45] (Table 2). Additionally, in BEACON trial, comparedwith the control, a triple regimen containing encorafenib, cetuximab, and a MEK inhibitor,
binimetinib also showed a significantly improved median OS (9.0 months in 224 subjects
vs. 5.4 months in 221 subjects) and confirmed RR (24% vs. 2%) [45] (Table 2). From theseresults, in 2020, the FDA-approved encorafenib for the treatment of mCRC with a BRAF
V600E mutation after prior therapy.
8. Concluding Remarks and Future Perspectives
This review focused on FDA-approved targeted drugs that are currently available totreat patients with mCRC. Our objective was to address the efficacy of these treatments.
We included most clinical trials that were associated with FDA approvals, reviewed trialassociated publications, and explored other relevant clinical trials.
Recent advancements in sequencing technologies have led to a better understanding ofcomprehensive genomic and proteomic alterations in mCRC, which helps to choose the correct treatment strategy. Although the latest therapeutic strategies have achieved substantial
progress in patients with mCRC, emerging resistance and lack of predictive biomarkers tocurrent targeted therapies remains a major problem in clinical settings. Further understanding of resistance mechanisms and exploration of biomarkers that can predict the treatment
sensitivity and efficacy/toxicity are required to test new evidence-based therapeutic strategies, expand treatment options, and realize personalized medicine [145–148].
To further improve the efficacy of treatments, innovations, such as CRC multicellular
3D models, patient-derived xenograft models, and single-cell sequencing strategy, andapplying new antibody-based therapies strategies, including chimeric antigen receptor
T cell therapy [149], antibody drug conjugates [150], radioimmunotherapy [151], andphotoimmunotherapy [152], may provide a survival benefit for patients with mCRC. To
date, we have developed several cancer-specific monoclonal antibodies, including antiEGFR antibodies using the CasMab method [153–163]. Currently, we are trying to further
develop these antibodies for the above-mentioned antibody-based therapy.

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Several promising targets including HER2, KRAS codon G12C mutation, and RET
have been revealed and their targeted therapies are under development and/or clinicalevaluation [164–166]. Continued comprehensive understanding of mCRC pathology andmolecular drivers, and developing novel therapeutic approaches are necessary to further
refine the treatment of mCRC. While validated targets, including RAS, BRAF, TRK, andMSI/dMMR, are important to choose the correct therapeutic strategy for patients with
mCRC [167]. This is because disclosure of additional targets may lead to the discovery ofpotential predictive biomarkers of treatment response and the correct selection of patients.
Further studies, including clinical trials, hold promise for improved outcomes and progress
toward personalized medicine for mCRC.
Author Contributions: Project design, Outline of the whole manuscript, T.O., M.K.K., Y.Y., A.T., Y.K.
and M.K.; Writing, T.O. Funding acquisition, Y.K., T.O. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was supported in part by Japan Agency for Medical Research and Development (AMED) under grant numbers JP21bm1004001 (to Y.K.), JP21am0401013 (to Y.K., T.O.),
JP22ama121008 (to Y.K.) and JP21am0101078 (to Y.K.) and by the Japan Society for the Promotionof Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) under grant numbers 18K08693(T.O.), 22K06783 (T.O.).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We would like to thank Akiko Harakawa, Shun-ichi Ohba, and Hiroyuki Inoue(Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation)
for their helpful suggestions. We also appreciate laboratory members for their contributing discussion.
Conflicts of Interest: The authors declare no conflict of interest.

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