Current status and future perspectives of bispecific antibodies in the treatment of lung cancer

Abstract

Monoclonal antibodies have been successfully incorporated into the current therapeutical landscape of lung cancer in the last decades. Recently, with technological advances, bispecific antibodies (bsAbs) have also shown robust efficacy in the treatment of malignant cancers, including lung cancer. These antibodies target two independent epitopes or antigens and have been extensively explored in translational and clinical studies in lung cancer. Here, we outline the mechanisms of action of bsAbs, related clinical data, ongoing clinical trials, and potent novel compounds of various types of bsAbs in clinical studies, especially in lung cancer. We also propose future directions for the clinical development of bsAbs, which might bring a new era of treatment for patients with lung cancer.

Introduction

Bispecific antibodies (bsAbs) are a class of antibodies designed to bind to two distinct epitopes or antigens and have recently been applied in the treatment of cancers, infections, and autoimmune diseases. The concept of bsAbs was first proposed by Nisonoff and Rivers^[1] in 1961, and Milstein and Cuello^[2] synthesized the first bsAb in 1983 via hybridoma technology. In the early period of this field, the production of bsAbs encountered several problems, such as immunogenicity, low production efficiency, and limited clinical application, hampering the development of this field.^[3] Nonetheless, with the advances in biotechnology, several novel bsAb-synthesis platforms, such as “Knob-Into-Hole,” “DuoBody,” “Crossmab,” and “TandAb,” have emerged.^[4-6] To date, hundreds of bsAbs have undergone evaluation in clinical trials worldwide. The dual specificity of bsAbs provides precise targeting and enhanced target-binding ability, compared with that of conventional antitumor drugs. Thus, relative to traditional chemotherapeutics, bsAbs have increased antitumor activity and reduced adverse effects and are associated with reduced treatment costs and incidence of drug resistance.

In the past decade, dramatic progress has been made in the use of bsAbs, such as blinatumomab and emicizumab, for the treatment of hematological malignancies.^[7,8] As for solid tumors, numerous bsAbs with promising results have been designed, albeit with a weaker antitumor effect than that in hematological malignancies. The progress of bsAbs in lung cancer is illustrated in [Figure 1]. To date, one bsAb—amivantamab—has been approved for the treatment of lung cancer, specifically for metastatic non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) exon 20 insertion (exon20ins) mutations, during or after platinum failure.^[9] Additionally, several others, such as KN046, M7824, SHR-1701, SI-B001, and AK112, have entered phase-III clinical trials for the treatment of lung cancer; and KN026, zenocutumab, and EMB-01 are at the early phase of clinical development. This review describes the mechanisms of action (MOAs) and clinical statuses of the bsAbs applied to the treatment of lung cancer.

Figure 1: Timeline of pivotal events of bsAbs in lung cancer. This timeline demonstrates the key concept of bsAbs in the treatment of lung cancer and the associated pivotal clinical trials. 1L: First-line; bsAb: Bispecific antibody; chemo: Chemotherapy; EGFR: Epidermal growth factor receptor; exon20ins: Exon 20 insertion; FDA: Food and Drug Administration; NRG1: Neuregulin 1; NSCLC: Non-small cell lung cancer; PFS: Progression-free survival.

MOAs

Currently, the MOAs of bsAbs in the treatment of lung cancer mainly include the following three categories: bridging immune cells with tumor cells for redirected cytotoxicity [Figure 2A], simultaneous blockade of two signaling pathways to inhibit tumor growth [Figure 2B], and targeting dual immunomodulatory molecules to promote immune responses [Figure 2C].

Figure 2: MOAs of bsAbs in the treatment of lung cancer. (A) One arm of a bsAb binds to TAA/TSA, and the other to a T-cell surface receptor, and this bridging mechanism redirects cytotoxicity. In addition, immune cells can be activated by targeting immune checkpoints. (B) bsAbs simultaneously target two TAAs, achieving antitumor effects by inhibiting two signaling pathways, inducing receptor degradation and internalization, and Fc-effector-mediated cytotoxicity. (C) bsAbs targeting dual immunomodulatory molecules may promote the cytotoxicity of T cells, enhance macrophage-mediated phagocytosis, and modify the immunosuppressive TME. ADCC: Antibody-dependent cell-mediated cytotoxicity; ADCP: Antibody-dependent cellular phagocytosis; AKT: Protein kinase B; bsAbs: Bispecific antibodies; CDC: Complement-dependent cytotoxicity; JAK: Janus kinase; MDSC: Myeloid-derived suppressor cell; MHC: Major histocompatibility complex; MOAs: Mechanisms of action; PD-1: Programed cell death protein 1; PD-L1: Programed death-ligand 1; PI3K: Phosphoinositide 3-kinase; RAF: Raf protein kinase; RAS: Rat sarcoma virus; SIRPα: Signal-regulatory protein alpha; sTAT: Signal transducer and activator of transcription; TAA: Tumor-associated antigen; TCR: T cell receptor; TME: Tumor microenvironment; Tregs: Regulatory T cells; TSA: Tumor-specific antigen.

Bridging immune cells with tumor cells for redirected cytotoxicity

The immune system performs immune surveillance. Through their T-cell receptors (TCRs), T cells can recognize and eliminate abnormal cells via detecting the cognate peptides presented by the major histocompatibility complex (MHC). However, tumor cells can escape immune surveillance by down-regulating the expression of MHC molecules, thereby rapidly proliferating in the body.^[10,11] Therefore, bsAbs can bypass MHC restriction by directly bridging T cells with tumor cells through simultaneous targeting of T-cell-associated antigens (CD3) and tumor-associated antigens (TAAs)/tumor-specific antigens (TSAs). In addition, immune synapses are formed between T cells and target cells, whereby the two cell types are brought in contact, and then the T cells are activated to secret perforin and other cytokines.^[12] In this situation, the bsAb enables T cells to reidentify and eliminate tumor cells. We also call this type of bsAbs bispecfic T-cell engagers (BiTEs). bsAbs targeting both programed cell death protein 1 (PD-1) and TAA also provide a similar bridging mechanism and are thus included in this sub-class. In addition, immune-checkpoint blockade activates immune cells, thereby increasing the cytotoxicity and antitumor activity.^[13] This sub-class includes AMG757 (delta-like protein 3 [DLL3] × CD3), BC3448 (EGFR × CD3), BI764532 (DLL3 × CD3), and IBI315 (PD-1 × human epidermal growth factor receptor-2 [HER2]).

Simultaneous blockade of two signaling pathways to inhibit tumor growth

Abnormal expression of receptor tyrosine kinases (RTKs) usually impairs the regulation of cell proliferation, resulting in tumorigenesis.^[14] RTK inhibitors targeting the ErbB family have become effective targeted antitumor drugs.^[15] However, tumor cells can develop drug resistance through compensatory signaling pathways, target mutations, or heterodimerization and crosstalk among the four members of the ErbB family.^[16] bsAbs blocking two RTK signaling pathways or two distinct epitopes can significantly inhibit the downstream signaling (e.g., the phosphoinositide 3-kinase [PI3K]-protein kinase B [AKT] and Rat sarcoma virus (RAS)-mitogen-activated protein kinase pathways) and induce receptor degradation and down-modulation, thus improving tumor clearance.^[17,18] bsAbs can also induce Fc-mediated killing of tumor cells via antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity.^[19,20] bsAbs with this MOAs comprise amivantamab (EGFR × c-mesenchymal-epithelial transition factor [c-MET]), SI-B001 (EGFR × human epidermal growth factor receptor-3 [HER3]), KN026 (HER2 × HER2), and zenocutumab (HER2 × HER3).

Targeting dual immunomodulatory molecules to promote immune responses

Immunomodulatory molecules, such as immune checkpoints, T-cell-costimulatory molecules, macrophage-coinhibitory molecules, and immune-suppressive molecules in the tumor microenvironment (TME), are involved in the regulation of immune functions. The use of bsAbs targeting dual immunomodulatory molecules is a promising therapeutic strategy in cancer. Immune checkpoints have immunosuppressive regulatory effects that facilitate tumor immune evasion.^[21] bsAbs that simultaneously target PD-1/programmed death-ligand 1 (PD-L1) and other immune checkpoints or immune-suppressive molecules can maximize T-cell antitumor activity by blocking more than one coinhibitory receptor or by modifying the immunosuppressive TME. Costimulatory molecules expressed on T cells can synergize with the TCR signaling to promote the proliferation and activation of T cells.^[22] bsAbs that combine immune-checkpoint blockade and stimulation of T-cell-costimulatory molecules also enhance the cytotoxic effect of T cells. Moreover, CD47, which belongs to macrophage-coinhibitory molecules, binds to the signal-regulatory protein alpha (SIRPα) receptor on macrophages to transmit a “do not eat me” signal.^[23] bsAbs simultaneously inhibiting the CD47/SIRPα and PD-1/PD-L1 pathways could enhance the cytotoxic effect of T cells and macrophage-mediated phagocytosis.^[24] The bsAbs acting in this way include KN046 (PD-L1 × cytotoxic T lymphocyte-associated antigen-4 [CTLA-4]), MGD013 (PD-1 × lymphocyte activation gene-3 [LAG3]), SHR-1701 (PD-L1 × transforming growth factor-beta [TGF-β]), GEN1046 (PD-L1 × 4-1BB), and IBI322 (PD-L1 × CD47).

Clinical Statuses of bsAbs in Lung Cancer

Targeted therapy guided by tumor-driving genes or immune-checkpoint blockade has become important arsenals in the treatment of advanced lung cancer. Unfortunately, the acquisition of drug resistance is inevitable in targeted therapy, and most patients do not respond to immunotherapy. Hence, a considerable number of bifunctional agents, such as bsAbs, are designed to address this issue. In this section, we will briefly review the activity of bsAbs in clinical trials. The bsAbs that have been granted expedited designations by Food and Drug Administration or evaluated in phase I–III clinical trials for the treatment of lung cancer are summarized in Table 1, and their targets are summarized in Figure 3.

Table 1 - bsAbs granted expedited designations by FDA or evaluated in phase I-III clinical trials.

MOAs	Targets	Drug	Company	Phase (NCT/CTR)	Regimen	Results
Simultaneous blockade of two signaling pathways to inhibit tumor growth
	EGFR × c-MET	Amivantamab	Janssen	Phase I (NCT02609776) [30,31]	Monotherapy	Approved
				Phase I/Ib (NCT04077463)	+ Lazertinib	ORR 36.0% (target population)
				Phase III (NCT04988295)	+ Lazertinib+ Chemo vs. Chemo	Ongoing
				Phase III (NCT04538664) [32]	+ Chemo vs. Chemo	Ongoing
				Phase III (NCT04487080) [33]	+ Lazertinib vs. Lazertinib vs. Osimertinib	Ongoing
	HER2 × HER3	Zenocutuzumab	Merus	Phase I/II (NCT02912949) [41]	Monotherapy	Fast track designation
	EGFR × HER3	SI-B001	Sichuan Baili Pharma.	Phase II (NCT05020457)	+ Chemo	DCR 87.5%
				Phase II/III (NCT05020769)	+ Osimertinib	Ongoing
	HER2 × HER2	KN026	Alphamab Oncology	Phase II (NCT04521179)	+ KN046	ORR 55.0% DCR 85.0% Grade ≥3 TRAEs 16.7%
Bridging immune cells with tumor cells for redirected cytotoxicity
	DLL3 × CD3	AMG757	Amgen	Phase I (NCT03319940) [74]	Monotherapy	DCR 43.0% Grade ≥3 TRAEs 25.0% Grade ≥3 CRS 2.0%
	PD-1 × HER2	IBI315	Innovent Biologics	Phase I (NCT04162327)	Monotherapy	ORR 20.0% DCR 40.0% No DLT
Targeting dual immunomodulatory molecules to promote immune responses
	PD-1 × VEGF-A	AK112	Akesobio	Phase I (NCT04047290) [69]	Monotherapy	ORR 23.5% Grade ≥3 TRAEs 10.3%
				Phase II (NCT04736823)	+ Chemo	ORR 76.9% (Cohort 1) ORR 68.4% (Cohort 2) ORR 40.0% (Cohort 3)
				Phase Ib/II (NCT04900363)	Monotherapy	ORR 60.0% (>10 mg/kg Q3W)
				Phase III (NCT05184712)	(AK112 vs. Placebo) + Chemo	Ongoing
	PD-L1 × CTLA-4	KN046	Alphamab oncology	Phase II (NCT04054531) [49]	+ Chemo	mPFS 5.9 months 12-month/15-month OS rate: 74.9%/74.9%
				Phase III (NCT04474119)	(KN046 vs. Placebo) + Chemo	Reaching the preset PFS endpoint
				Phase II/III (NCT05001724)	+ Lenvatinib vs. Docetaxel	Ongoing
	PD-1 × CTLA-4	AK104	Akesobio	Phase Ib/II (NCT04646330) [51]	+ Anlotinib	ORR 70.6% Grade ≥3 AK104-related TEAEs 14.3%/5.9% (immunotherapy-naïve/resistant cohort)
	PD-L1 × TGF-β	M7824	Merck KGaA	Phase III (NCT03631706)	M7824 vs. Pembrolizumab	Failed
	PD-L1 × TGF-β	SHR-1701	Jiangsu HengRui Medicine	Phase I (NCT03710265) [65]	Monotherapy	ORR 17.8% Grade ≥3 TRAEs 18.4%
				Phase I (NCT03774979) [66]	Monotherapy	ORR 36.5% DCR 67.3% Grade = 3 TRAEs 21.1%
				Phase III (NCT05132413)	(SHR-1701 vs. Placebo) + Chemo ± Bevacizumab	Ongoing
	PD-L1 × TGF-β	PM8001	BIOTHEUS	Phase I/IIa (ChiCTR2000033828)	Monotherapy	ORR 10.4% Grade ≥3 TRAEs 16.7%
	PD-L1 × 4-1BB	GEN1046	Genmab	Phase II (NCT03917381) [59]	Monotherapy	ORR 6.5% Grade 3–4 TRAEs 27.9%
	PD-1 × 4-1BB	IBI319	Innovent Biologics	Phase I (NCT04708210)	Monotherapy	No MTD, DLT TRAEs 52.4%
	PD-L1 × CD47	IBI322	Innovent Biologics	Phase I (NCT04328831)	Monotherapy	ORR 33.3% Grade ≥3 TRAEs 22.4%
	PD-1 × CD47	HX009	Hanxbio	Phase II (NCT04886271) [72]	Monotherapy	TRAEs 47.6% No DLT
	PD-1 × LAG3	MGD013	ZLAB	Phase I (NCT03219268) [54]	Monotherapy	No MTD Grade ≥3 TRAEs 23.2%
	PD-L1 × TIM3	LY3415244	Eli Lilly and Company	Phase I (NCT03752177) [57]	Monotherapy	Terminated due to ADA

ADA: Anti-drug antibodies; bsAbs: Bispecific antibodies; Chemo: Chemotherapy; c-MET: c-mesenchymal-epithelial transition factor; CRS: Cytokine release syndrome; CTLA-4: Cytotoxic T lymphocyte-associated antigen-4; CTR: Clinical Trial Registry; DCR: Disease control rate; DLL3: Delta-like protein 3; DLT: Dose limiting toxicity; EGFR: Epidermal growth factor receptor; FDA: Food and Drug Administration; HER2: Human epidermal growth factor receptor-2; HER3: Human epidermal growth factor receptor-3; LAG3: Lymphocyte activation gene-3; MOAs: Mechanisms of action; mPFS: Median progression-free survival; MTD: Maximum tolerated dose; NCT: National Clinical Trial; ORR: Objective response rate; OS: Overall survival; PD-1: Programed cell death protein 1; PD-L1: Programed death-ligand 1; PFS: Progression-free survival; Pharm.: Pharmaceutical; TEAEs: Treatment-emergent adverse events; TGF-β: Transforming growth factor-beta; TIM3: T cell immunoglobulin and mucin-domain containing-3; TRAEs: Treatment-related adverse events; VEGF-A: Vascular endothelial growth factor A.

Figure 3: Targets of bsAbs in lung cancer. This figure summarizes the targets of bsAbs in lung cancer. EGFR, c-MET, HER3, and HER2 are TAAs in NSCLC. DLL3 is a TSA in SCLC. LAG3, 4-1BB, CTLA-4, TIM3, and TIGIT are T-cell costimulation/coinhibition molecules. CD47 belongs to macrophage coinhibitory molecules. VEGF-A and TGF-β are immune-suppressive molecules in the TME. bsAbs: Bispecific antibodies; c-MET: c-mesenchymal-epithelial transition factor; CTLA-4: Cytotoxic T lymphocyte-associated antigen-4; DLL3: Delta-like protein 3; EGFR: Epidermal growth factor receptor; HER2: Human epidermal growth factor receptor-2; HER3: Human epidermal growth factor receptor-3; LAG3: Lymphocyte activation gene-3; NSCLC: Non-small cell lung cancer; PD-1: Programed cell death protein 1; PD-L1: Programed death-ligand 1; SCLC: Small cell lung cancer; TAAs: Tumor-associated antigens; TGF-β: Transforming growth factor-beta; TIGIT: T cell immunoreceptor with immunoglobulin (Ig) and ITIM domains; TIM3: T cell immunoglobulin and mucin-domain containing-3; TME: Tumor microenvironment; TSA: Tumor-specific antigen; VEGF-A: Vascular endothelial growth factor A.

bsAbs for targeted therapy

Acquisition of resistance to targeted therapy mainly involves activation of intracellular signals through homologous or heterologous dimers between RTKs and activation of compensatory signaling pathways.^[16] To solve the problem of drug resistance, researchers and clinicians are constantly searching for new targets, and developing new-generation targeted drugs and combinatorial treatments. bsAbs in the treatment of lung cancer mainly target the ErbB RTK family members, which comprise EGFR, HER2, HER3, and human epidermal growth factor receptor-4, and participate in overlapping downstream signaling pathways.^[25] bsAbs that simultaneously target two independent epitopes or antigens can effectively inhibit downstream oncogenic signaling and thus efficiently suppress tumor growth.

EGFR × c-MET

Approximately 40% of NSCLC patients in Asia harbor an EGFR mutation.^[26] Several EGFR-tyrosine kinase inhibitors (TKIs) have achieved excellent clinical outcomes in this population. However, the acquisition of drug resistance is inevitable, and the subsequent treatment is limited, resulting in a poor prognosis. The resistance mechanisms include, but are not limited to, the emergence of the EGFR T790M mutation, EGFR C797S mutation, abnormal cellular-mesenchymal epithelial transition factor (c-MET) activity, and RAS mutations.^[27] The EGFR and c-MET signaling pathways are partially compensatory. Extensive evidence has demonstrated crosstalk between EGFR and c-MET in lung cancer, including EGFR-dependent phosphorylation and EGFR-ligand-induced c-MET activation.^[28,29] Therefore, bsAbs co-targeting EGFR and c-MET may prove effective in the treatment of EGFR-positive lung cancer.

Amivantamab

Amivantamab is an anti-EGFR × c-MET bsAb developed by Jassen Biotech, filling the gap in standard targeted therapy for metastatic NSCLC with EGFR exon20ins mutations. In a phase-I study of amivantamab in patients with local or metastatic NSCLC who had progressed or already underwent platinum-based chemotherapy (CHRYSALIS, NCT02609776), the EGFR exon20ins mutant cohort reported an objective response rate (ORR) of 40.0% and a median progression-free survival (mPFS) of 8.3 months (n = 81).^[30] Based on these results, FDA granted accelerated and conditional approval to amivantamab in 2021 for metastatic NSCLC with EGFR exon20ins mutations during or after platinum failure.^[9]

Apart from effectively solving the problem of poor survival in NSCLC harboring non-classical EGFR mutations, amivantamab also offers new potential strategies for the treatment of osimertinib-resistant NSCLC patients. Another set of results from the CHRYSALIS study on the combinatorial application of amivantamab with lazertinib demonstrated an ORR of 36% in the post-osimertinib, chemo-naive setting.^[31] Currently, in a phase-Ib trial, CHRYSALIS-2 is evaluating the efficacy of amivantamab + lazertinib in post-osimertinib and -platinum EGFR-mutant NSCLC (NCT04077463) in its cohort A and updated its results at American Society of Clinical Oncology (ASCO) 2022. The ORR was 36% with 1 complete response (CR) and 17 partial responses (PRs) in the evaluable target population (n = 50). In a more heavily-pretreated population (prior treatment lines >3, n = 56), the ORR was 29% with 1 CR and 15 PRs. The most common adverse events in the overall population were infusion-related reactions (IRR) (65%), paronychia (49%), rash (41%), and stomatitis (39%). Based on the existing data, no significant difference has been observed between amivantamab + lazertinib treatment with chemotherapy and without chemotherapy in post-osimertinib setting. Moreover, a phase-III trial of combinatorial application of amivantamab and lazertinib along with chemotherapy vs. chemotherapy alone in NSCLC patients after osimertinib failure (NCT04988295) has been designed to verify the effect of the combinatorial therapy in overcoming osimertinib resistance.

Based on the above promising results of amivantamb as a second-line (2L) therapy, clinical trials of amivantamab as a first-line (1L) therapy are currently in progress. Phase III trial of amivantamab in combination with chemotherapy vs. chemotherapy alone in NSCLC patients harboring EGFR exon20ins mutations (NCT04538664)^[32] and Phase III trial of amivantamab + lazertinib vs. lazertinib or osimertinib alone in advanced EGFR-mutant NSCLC (NCT04487080)^[33] are in progress. However, no preliminary results have been disclosed yet. Other ongoing clinical trials of amivantamab focus on combinatorial therapies in NSCLC (NCT04965090, NCT05299125, and NCT04085315) or monotherapy in advanced solid tumors (NCT04606381).

EGFR × HER3

Different from the other ErbB family members, HER3 lacks an intrinsic kinase activity, and thus there are not many studies on HER3-targeted drugs. However, HER3 can directly activate the PI3K/AKT signaling pathway through its intracellular sites and can also heterodimerize with EGFR/HER2 to activate downstream oncogenic signaling. Hence, HER3 overexpression in lung cancer can promote EGFR TKI drug resistance.^[34,35]

SI-B001

SI-B001 is a dual-targeting EGFR × HER3 bsAb developed by Sichuan Baili Pharmaceutical. A phase-II study of SI-B001 combined with chemotherapy has been conducted in locally advanced or metastatic EGFR/anaplastic lymphoma kinase (ALK) wild-type (WT) NSCLC patients who progressed on prior anti-PD-1 treatment with or without post-platinum chemotherapy (NCT05020457). The preliminary results revealed by Baili Pharmaceutical indicate that 49 subjects were enrolled and yielded a disease control rate (DCR) of 87.5% (7/8), of which one case achieved PR. Another phase II/III trial has been conducted to investigate the safety and efficacy of SI-B001 + osimertinib in patients with locally advanced or metastatic NSCLC patients who progressed on previous EGFR-TKI treatment or with non-TKI-sensitizing mutations or patients with an EGFR exon20ins mutation (NCT05020769). With limited available data, it is currently hard to foresee the future applications and effects of SI-B001, and we hope to hear additional encouraging news from such studies.

HER2 × HER2

HER2 mutation appears in approximately 2% of NSCLC. HER2 is activated by other members of the ErbB family and is the preferred heterodimerization partner of all ErbB members. At present, there is no sufficient clinical evidence to prove that NSCLC with HER2 expression benefits from the existing HER2-targeted therapies.^[36] This is probably due to that differing from HER2-mutant breast cancer or gastric cancer, NSCLC mainly obtained a HER exon20ins mutation.^[37] It is an unmet need to seek out new targeted therapy for HER2-mutant NSCLC. bsAbs targeting different HER2 epitopes may deliver an enhanced blockade of downstream signaling, and thus inhibition of dimerization may offer a new treatment option in HER2-positive NSCLC.

KN026

KN026 is a bsAb that binds to two distinct HER2 epitopes (extracellular domains II and IV), attaining an enhanced blockade of the HER2 signaling pathway compared with anti-HER2 monoclonal antibodies (mAbs). A phase-II clinical trial is currently in progress to evaluate the safety, efficacy, and tolerability of KN026 in combination with KN046 in locally advanced HER2-positive solid tumors (NCT04521179). The results were published at American Association for Cancer Research (AACR) 2022 and showed an ORR of 55%, DCR of 85%, and 6-month progression-free survival (PFS) rate of 84.1% in the overall population (n = 24), in which four HER2-mutant NSCLC patients were enrolled. The grade ≥3 treatment-related adverse events (TRAEs) occurred in 16.7% of the patients. The most common TRAEs were IRR (29.2%), diarrhea (19.4%), and elevated alanine aminotransferase (ALT, 16.7%). Despite the promising clinical results, the role of KN026 per se in HER2-mutant NSCLC is not certain due to the combinatorial nature of the study and the small sample size. Thus, additional clinical trials are needed to prove the efficacy of KN026.

HER2 × HER3

HER2 × HER3 dimerization is regarded as the most robust heterodimerization in the EGFR family. Additionally, neuregulin 1 (NRG1) fusion can bind to HER3, promoting heterodimerization of HER3 with HER2, thereby inducing cell proliferation and tumorigenesis.^[38] The NRG1 fusion gene is a rare oncogene driver, occurring in <1% of NSCLC and typically causes a poor response to standard chemotherapy, single-agent immune-checkpoint inhibitors (ICIs), or a combination of both.^[39,40] Hence, bsAbs simultaneously binding to HER2 and HER3 on the tumor cell surface may be effective in the treatment of patients with NRG1 fusion mutations.

Zenocutuzumab

Zenocutuzumab is a dual-target HER2 × HER3 bsAb with strong ADCC activity. It prevents downstream signal transduction by interfering with NRG1-binding to HER3 and inhibiting HER2:HER3 dimerization. In the early proof-of-concept stage, a stage IIIB NSCLC patient with CD74-NRG1 fusion mutation, who had progressed on previous therapy, obtained PR with a tumor volume reduction of 33%, and tumor shrinkage in the brain metastases after receiving zenocutuzumab intravenously (IV) 750 mg once every two weeks (Q2W).^[20] In 2021, zenocutuzumab was granted fast-track designation in NRG1+ metastatic solid tumors by FDA. A phase I/II trial involving 18 NSCLC patients is currently assessing the effect of zenocutumab in patients with solid tumors harboring an NRG1 fusion (NCT02912949). In the overall population, the preliminary results of the study demonstrated that 25 patients had tumor regression, and 9 of 33 patients had a confirmed response with an ORR of 27%. No obvious cardiotoxicity was observed, and no patient required dose reduction due to toxicity.^[41]

bsAbs for Immunotherapy

The development of PD-(L)1 inhibitors constitutes a critical breakthrough in lung cancer treatment. However, the response rate to monotherapy via ICIs is low, and the acquisition of drug resistance is inevitable. Several targets, such as immune checkpoints, T-cell-costimulatory factors, macrophage-coinhibitory factors, and immune-suppressive factors in the TME, are ideal for promoting immune responses. bsAbs targeting dual immunomodulatory molecules or bispecfic T-cell engagers are promising to further improve the efficacy of immunotherapy. These two types of bsAbs are currently widely evaluated for lung cancer immunotherapy in clinical trials.

bsAbs targeting dual immunomodulatory molecules

Dual immune-checkpoint blockade

PD-(L)1 × CTLA-4

CTLA-4 belongs to the immunoglobulin superfamily (igSF) and is mainly expressed on the surface of activated T lymphocytes. CTLA-4 shares approximately 30% homology with the T-cell-costimulatory molecule CD28, competitively binding to the same ligands CD80 (B7-1)/CD86 (B7-2) with higher affinity. Then the binding delivers a negative signal to inhibit the proliferation and activation of T cells.^[42-44] CTLA-4 is an effective target for the treatment of lung cancer. The combinatorial therapy of ipilimumab (anti-CTLA-4 monoclonal antibody [mAb]) with nivolumab (anti-PD-1 mAb) in patients with NSCLC has been approved by the FDA.^[45] The preliminary results from the clinical trials on bsAbs (including KN046 and AK104) that bind to PD-(L)1 and CTLA-4 are promising.

KN046: KN046 is a novel recombinant humanized anti-PD-L1/CTLA-4 bsAb with high PD-L1 and low CTLA-4 affinity. In addition, the WT immunoglobulin G1 (IgG1) Fc portion of KN046 results in regulatory T cell (Treg) depletion.^[46] In a phase-II study of KN046 as a second-line treatment in patients with metastatic NSCLC (NCT03838848, KN046-201), the mPFS was 3.68 months. Longer mPFS (7.29 months) has been reported in patients with lung squamous cell carcinoma (LSCC). KN046 had longer mPFS than nivolumab in the second-line treatment of LSCC in a phase-III Checkmate-017 study (7.29 months vs. 3.50 months^[47]). However, outcomes from such a single-arm trial with a small sample size are not quite reliable. A total of 24 of the 64 patients (37.5%) experienced grade ≥3 TRAEs.^[48] In addition, a phase-II/III trial of KN046 combined with lenvatinib vs. docetaxel in subjects with NSCLC after failure of anti-PD-(L)1 agent (NCT05001724) is ongoing.

In the field of first-line treatment, a phase-II study of KN046 combined with chemotherapy in patients with advanced NSCLC (NCT04054531) showed that among 81 efficacy-evaluable patients, the mPFS was 5.9 months, and the 12-month and 15-month overall survival (OS) rates were both 74.9%. The mPFS of LSCC patients with the PD-L1 expression level of ≥1% reached 10.8 months.^[49] Furthermore, the first interim analysis of the phase III study of KN046 combined with chemotherapy as a first-line treatment of LSCC (NCT04474119) was successfully completed in April 2022, reaching the preset primary endpoint of PFS. KN046 combined with chemotherapy has shown outstanding antitumor activity, and we look forward to the PFS and OS data with longer follow-up than Checkmate-9LA and Keynote-407. The results from the phase-III study may change the treatment landscape for advanced NSCLC.

AK104: AK104 is an anti-PD-1 × CTLA-4 bsAb with a symmetric structure of tetravalent IgG1-ScFv developed by the unique Tetrabody technology of Kangfang Biotech. In the phase Ia dose-escalating study of AK104 in Australia (NCT03261011), among 25 patients with solid tumors, the ORR and DCR were 24% and 44%, respectively.^[50] In addition, a phase Ib/II trial of AK104 in combination with anlotinib in advanced NSCLC (NCT04646330) has been reported. The ORR in immunotherapy-naïve NSCLC patients was 70.6%. Six patients who progressed on prior ICIs were enrolled with DCR of 100% and ORR of 16.7%. The incidence of grade ≥3 AK104-related treatment-emergent adverse events was 14.3% in the immunotherapy-naïve cohort and 5.9% in the immunotherapy-resistant cohort.^[51]

PD-1 × LAG3

LAG3 is a type-I transmembrane protein of the igSF with homology to CD4. LAG3 expression is mainly observed in activated T cells, natural killer (NK) cells, and Tregs cells, as well as in plasmacytoid dendritic cells (DCs) and B cells. LAG3 binds to MHC-II, its key ligand, thereby suppressing T-cell proliferation and activation. It has also been reported that LAG3 may inhibit the suppressive function of Tregs.^[52] A recent study using multiplexed quantitative immunofluorescence reported that LAG3 is expressed on tumor-infiltrating lymphocytes in 41.5% of NSCLC cases.^[53] Further, LAG3 was associated with poor survival, thus indicating that LAG3 may be an ideal therapeutic target for lung cancer.

MGD013: MGD013, known as a LAG3 and PD-1 dual-affinity re-targeting protein, has been evaluated in patients with unresectable and metastatic tumors in a first-in-human, open-label, dose-escalation phase I study (NCT03219268). This bsAb was well tolerated and no maximum tolerance dose (MTD) was defined. The incidence of grade ≥3 TRAEs was 23.2%, and the most common TRAEs were fatigue and nausea.^[54] We hope to see additional evidence of antitumor activity in the near future.

PD-L1 × TIM3

T cell immunoglobulin and mucin-domain containing-3 (TIM3) is a member of the TIM family of immunoregulatory proteins, originally identified to be expressed on interferon-γ (IFN-γ)-producing CD4⁺ and CD8⁺ T cells. Subsequently, Treg cells, NK cells, and myeloid cells have been demonstrated to express TIM3.^[52,55] The ligands of LAG3 include galectin-9, high-mobility group protein B1 (HMGB1), carcinoembryonic antigen cell adhesion molecule 1 (CEACAM-1), and phosphatidyl serine (PtdSer). The interaction of TIM3 with its ligands exhausts immune cells.^[56] TIM3 expression has been detected in 25.3% of NSCLC cases and is related to poor prognosis.^[53] Therefore, concurrently blocking PD-(L)1 and TIM3 may be a feasible therapeutic option.

LY3415244: A phase I study of LY3415244 (anti-PD-L1 × TIM3 bsAb) in patients with advanced solid tumors (NCT03752177) was terminated early due to the development of anti-drug antibodies (ADA) in all the patients. The high ADA titers negatively influenced soluble TIM3 target engagement.^[57] The study underscores the need for early and comprehensive immunogenicity risk assessment, especially when developing bsAb structures.

Targeting T-cell-costimulatory molecules and PD-(L)1

PD-(L)1 × 4-1BB

4-1BB is a member of the tumor necrosis factor receptor superfamily. It is mainly expressed on activated CD4⁺ and CD8⁺ T cells, as well as on activated NK cells, NK T cells, Tregs, DCs, and other myeloid-lineage cells. The interaction of 4-1BB and its ligands (4-1BBL) can enhance the function of CD8⁺ T cells and stimulate DCs and macrophages to improve antitumor immune responses. 4-1BB activation has also been reported to promote ADCC effect induced by NK cells.^[22,58] bsAbs that block PD-(L)1 inhibitory pathway and conditional 4-1BB stimulation are viable therapeutic strategies in solid tumors.

GEN1046: GEN1046 is a full-length IgG1 PD-L1 × 4-1BB bsAb developed via the DuoBody technology platform. The efficacy and safety results in a phase-I/IIa study of GEN1046 in patients with advanced solid tumors (NCT03917381) have recently been reported. Patients with the following common tumor types were enrolled: colorectal cancer (12 cases), ovarian cancer (9 cases), pancreatic cancer (6 cases), and NSCLC (6 cases). Among 61 patients, the ORR and DCR were 6.5% and 65.6%, respectively, and PR was observed in two patients with NSCLC. GEN1046 was reported to have a manageable safety profile with early evidence of antitumor activity. Most TRAEs were grade 1–2 and 17 (27.9%) patients experienced grade 3–4 TRAEs.^[59] No grade ≥4 transaminase elevation or treatment-related bilirubin elevation was observed. Compared with anti-4-1BB mAbs, GEN1046 has significantly reduced hepatotoxicity. Its efficacy in the treatment of lung cancer needs to be verified with additional clinical data.

IBI319: IBI319, a bsAb blocking PD-1 while activating 4-1BB efficiently restricts T-cell activation to the TME and has demonstrated efficient tumor control and reduced liver toxicity in preclinical studies.^[60] At ASCO 2022, the preliminary outcomes of a phase-I study of IBI319 in advanced malignant tumors (NCT04708210) have been reported. Among the 21 enrolled patients, no dose-limiting toxicity (DLT) was observed up to 6.0 mg/kg, and dose escalation is still in progress. The incidence of TRAEs was 52.4% and grade ≥3 TRAEs were not observed. More importantly, IBI319 had a lack of hepatotoxicity, which frequently occurs in the application of anti-4-1BB antibodies.

Targeting immune-suppressive molecules in TME and PD-(L)1

PD-L1 × TGF-β

TGF-β belongs to a superfamily of cytokines and plays an important role in regulating the immune microenvironment with potent immunosuppressive effects on both innate and adaptive immune cells, including CD4⁺ and CD8⁺ T cells, DCs, macrophages, and NK cells.^[61] Many studies have identified the overexpression of TGF-β in various types of tumors, and TGF-β expression can significantly predict the poor prognosis in patients with lung cancer.^[62] BsAbs targeting PD-L1 × TGF-β present a viable strategy for the treatment of lung cancer.

M7824: M7824, a dual anti-PD-L1 and TGF-β trap molecule, has been tested in a phase-I study as a second-line treatment in advanced NSCLC (NCT02517398). The ORR was 36.0% and 85.7% in the PD-L1-positive and high PD-L1-expressing populations, respectively.^[63] The efficacy of M7824 as a second-line treatment of NSCLC is surprising. However, the phase-III study of M7824 vs. pembrolizumab in the first-line treatment of patients with stage IV NSCLC with high PD-L1 expression (NCT03631706) has been analyzed by an independent data-monitoring committee and found to be unlikely to meet the primary endpoint of PFS, and thus the study was terminated in January 2021.^[64] The failure of the phase III study of M7824 suggests that we cannot over-interpret the results from single-arm trials.

SHR-1701: SHR-1701 is the first domestic anti-PD-L1 × TGF-β bsAb to enter the clinical stage. A phase-I study aimed to determine the safety profile, MTD, and recommended phase-II dose (RP2D) of SHR-1701 in refractory solid tumors (NCT03710265). The ORR was 17.8%, of which eight cases achieved PR, and two cases were lung adenocarcinoma. The DCR was 40.0%, and the incidence of grade ≥3 TRAEs was 18.4%. MTD was not reached, and 30 mg/kg once every 3 weeks (Q3W) was recommended as the RP2D.^[65] Moreover, the outcomes of SHR-1701 as a first-line therapy for PD-L1⁺ advanced/metastatic NSCLC from a clinical expansion cohort of a phase I study (NCT03774979) were reported. The ORR and DCR were 36.5% and 67.3%, respectively. The incidence of grade-3 TRAEs was 21.1%, and the most common TRAEs were rash, anemia, decreased appetite, hyperthyroidism, hypothyroidism, and increased ALT.^[66] SHR-1701 showed a manageable safety profile and encouraging antitumor activity in patients with advanced NSCLC. A phase-III study of SHR-1701 + bevacizumab and chemotherapy in NSCLC (NCT05132413) and other phase-II trials of SHR-1701 in lung cancer (NCT04884009, NCT04580498, NCT04699968, and NCT05177497) are underway.

PM8001: PM8001 is a bsAb targeting PD-L1 × TGF-β. This bsAb has a small molecular weight and thus can easily infiltrate solid tumors. A phase I/IIa study of PM8001 in patients with advanced solid tumor (ChiCTR2000033828) has presented preliminary results at ASCO 2022. Among 67 efficacy-evaluable patients, the ORR and DCR were 10.4% and 53.7%, respectively. Of the total 11 patients with PRs, four had received anti-PD-1 therapy. Grade ≥3 TRAEs occurred in 16.7% of the patients, and the most common grade ≥3 TRAEs were anemia and rash. Further phase-I trial of PM8001 in combination with other anti-tumor agents in lung cancer (ChiCTR2100052617) is currently ongoing.

PD-1 × VEGF-A

Vascular endothelial growth factor A (VEGF-A) is widely expressed in almost all solid tumors and is considered to play a key role in tumor angiogenesis.^[67] Studies have corroborated that VEGF-A induces an immunosuppressive microenvironment through inhibiting DC maturation, accumulating myeloid-derived suppressor cell, and inducing Treg cells. Targeting VEGF-A enhances inhibition of PD-1 as well as decreases PD-1 expression on intratumoral CD8⁺ T cells.^[68] These observations suggest that anti PD-1 therapy in combination with antiangiogenic therapy in antitumor therapy may be effective. Hence, researchers have focused on bsAb targeting both PD-1 and VEGF-A, such as AK112.

AK112: AK112 is a bsAb targeting both PD-1 × VEGF and has been developed by Akeso. AK112 was first evaluated in a phase-I study in advanced solid tumors resistant/refractory to standard therapies (NCT04047290). In the 17 efficacy-evaluable patients at dose levels ≥3 mg/kg Q2W, the ORR was 23.5% and DCR was 64.7%. The incidence of grade ≥3 TRAEs were 10.3%, and the treatment-related serious adverse event (SAE) occurred in one patient (3.4%).^[69] The latest results of the phase-II trials of AK112 monotherapy (AK112-202, NCT04900363) and AK112 combined with chemotherapy (AK112-201, NCT04736823) in patients with advanced NSCLC were subsequently announced at the ASCO 2022. There were a total of three cohorts in part 1 of AK112-201. Treatment-naïve population with WT EGFR/ALK (Cohort 1) reported an ORR of 76.9% and a DCR of 100% (n = 26). The EGFR-mutant population who had failed prior EGFR-TKI therapies (Cohort 2) reported an ORR of 68.4% and a DCR of 94.7% (n = 19). The patients who progressed after anti PD-(L)1 and chemotherapy (Cohort 3) received an ORR of 40% and a DCR of 80% (n = 20). In the overall population, grade ≥3 AEs occurred in 28.6% of the patients, including two deaths. AK112 monotherapy in advanced NSCLC was well tolerated and demonstrated superior antitumor activity in treatment-naïve patients with PD-L1 tumor proportion score ≥1%. A total of 35 patients who received doses >10 mg/kg Q3W had an ORR of 60.0% and a DCR of 97.1%. The most common TRAEs were proteinuria (17.0%), hypertension (16.0%), increased lipase (12.8%), and increased ALT (12.8%); and the incidence of grade ≥3 TRAEs was 10.6%. Based on the promising results from the phase-II exploratory trials, a phase-III study of AK112 or placebo combined with chemotherapy in patients with EGFR-mutant non-squamous NSCLC who failed EGFR-TKI treatment (NCT05184712) and other phase I/II trials of AK112 in lung cancer (NCT05116007 and NCT05247684) are ongoing.

Targeting macrophage-coinhibitory molecules and PD-(L)1

PD-(L)1 × CD47

CD47 is a cell surface protein in the igSF and is normally expressed in healthy cells at low level and overexpressed in tumor cells, which binds to SIRPα receptor on myeloid cells to deliver a “do not eat me” signal.^[23] Blocking the CD47-SIRPα axis is currently considered a potential approach to cancer therapy via increasing phagocytosis of tumor cells by macrophages. However, healthy cells, such as erythrocytes, may be impaired by anti-CD47 mAbs.^[70] Furthermore, several bsAbs targeting PD-(L)1 × CD47 are currently tested in clinical trials and thought to maximize the efficacy of CD47 blockade with minimal interference with red blood cells.

IBI322: IBI322 not only inhibits the PD-1/PD-L1 pathway to enhance the cytotoxicity of T cells, but also blocks the SIRPα/CD47 axis to promote macrophage phagocytosis.^[71] A phase-I study of IBI322 (an anti-PD-L1 × CD47 bsAb) monotherapy in patients with advanced malignancies who failed standard therapy (NCT04328831) presented preliminary results at AACR 2022. Among nine patients with NSCLC (including four patients who had failed prior ICI therapy) at an effective dose (≥10 mg/kg), three (33.3%) cases achieved PR, and five (55.6%) cases achieved stable disease (SD). Grade ≥3 TRAEs occurred in 13 (22.4%) patients, and the most common grade ≥3 TRAE was thrombocytopenia (12.1%). All the thrombocytopenia cases were asymptomatic, transient, and resolved within 10 days. A phase-II study of IBI322 + bevacizumab and chemotherapy in ALK-rearranged NSCLC (NCT05296278) and phase-II trial of IBI322 + lenvatinib in small cell lung cancer (SCLC) (NCT05296603) are ongoing.

HX009: HX009 is an anti-PD-1 × CD47 bsAb that simultaneously activate innate and acquired immune responses for a synergistic anti-tumor effect. The preliminary results of a phase-I dose escalation study of HX009 in patients with advanced malignancies (NCT04886271) have been reported. There were seven dose levels designed (0.1, 0.3, 1 , 2, 3, 5, and 7.5 mg/kg). HX009 was well tolerated in 10 (47.6%) patients experiencing TRAEs, and no DLT was observed up to 7.5 mg/kg.^[72] At the 1 and 5 mg/kg cohorts, the antitumor activity of HX009 was seen, and the corresponding phase-II trial is ongoing.

BiTEs

As mentioned above, various bsAbs have good application prospects in NSCLC. However, there are fewer bsAbs developed for the treatment of SCLC than those developed against NSCLC. To date, only BiTEs targeting DLL3 have achieved significant progress in the treatment of SCLC. Hence, in this section, we will discuss the use of BiTEs for the treatment of SCLC and NSCLC.

BiTEs for SCLC

DLL3 × CD3

DLL3 is an inhibitory notch ligand and functions in various cellular processes, including differentiation, proliferation, survival, and apoptosis. It is overexpressed in >85% of SCLC cases and is a highly tumor-selective cell-surface target with rare expression in healthy tissues.^[73] Thus, DLL3 is an appealing therapeutic target in SCLC. The previous DLL3-targeted antibody-drug conjugate, rovalpituzumab tesirinea, has not shown the expected efficacy. However, bsAbs designed to simultaneously target DLL3 and CD3 have demonstrated promising results, redirecting T-cell cytotoxicity to DLL3-expressing tumor cells.

AMG757: AMG757 is a DLL3-targeting half-life extended bispecific T-cell engager designed to bind to DLL3 on cancer cells and CD3 on T cells. In a phase-I trial involving 64 SCLC patients with 28 patients (44%) who received prior anti-PD-1/PD-L1 therapy, AMG757 treatment of SCLC (NCT03319940) had a confirmed PR in 13% of the patients, and the DCR was 43%. It remained tolerable at doses up to 100 mg and attained an unconfirmed PR in 6/8 patients (63%). Cytokine release syndrome (CRS) was the most common TRAE, which was reported in 27 patients (43%), with 1 patient (2%) having a grade ≥3 CRS. Unfortunately, one patient (2%) had grade-5 pneumonitis.^[74] The effect of AMG757 in the phase I trial is not impressive, which may be related to the fact that approximately half of the enrolled patients did not benefit from immunotherapy. Increasing the dose may improve the efficacy of AMG757, and the optimal dose remains to be explored. Additionally, one patient died due to pneumonia, and the dose she received was not high, requiring continued exploration of the safe doses and associated risk factors. Based on these results, a phase-II study is evaluating AMG 757 administered in patients with recurrent/refractory SCLC after two or more prior lines of treatment (NCT05060016). To further enhance the efficacy of AMG757, phase-I trials of combinatorial therapies are in progress, such as AMG757 combined with AMG404 (NCT04885998),^[75] or with an ICI or chemotherapy (NCT05361395).

BiTEs for NSCLC

PD-1 × HER2

Currently, several reports have verified that anti-PD-1 mAb can be a potential therapeutic partner of anti-HER2 mAb in the treatment of HER2-positive tumors.^[76] Hence, bsAbs that simultaneously bind to HER2 and PD-1 may deliver a synergistic effect while bridging T cells to tumor cells for achieving tumor localization. Immune synapses are formed between the tumor cell and immune cell to mediate immune-cell cytotoxicity, bypassing antigen presentation.

IBI315: IBI315 is a bsAb targeting HER2 and PD-1 and has shown encouraging antitumor ability in preclinical studies. In an open-label, multicenter phase-I study, the effect of IBI315 was assessed in patients with advanced solid tumors expressing HER2 (IHC1⁺ or higher) who had failed/were intolerant or refused standard therapy (NCT04162327). The results were revealed at the Chinese Society of Clinical Oncology 2021; the ORR and DCR were 20% and 40%, respectively, among the 15 patients with evaluable efficacy. Acceptable tolerance was observed, and no DLT occurred at the dose level of 15 mg/kg Q3W.

Potential Candidates

Apart from the above-mentioned bsAbs, several other bsAbs have also shown promising preclinical results and are currently in early clinical development [Table 2]. As to lung cancer with driver gene mutations, EMB-01 and MCLA-129, which are two other anti-EGFR × MET bsAbs, have shown promising results in preclinical studies on antitumor activity.^[77] EMB-01 has demonstrated superior antitumor activity to EGFR and MET inhibitors used alone or in combination, even in an EGFR drug resistance model.^[78] Similarly, the growth of NSCLC cell lines with mutations of EGFR L868R, T790M, or EGFR19 del, c-MET amplified was significantly inhibited by MCLA-129. Importantly, MCLA-129 could overcome the hepatocyte growth factor (HRG)-mediated EGFR TKI resistance mechanism.^[79] Both of these bsAbs are under investigation in phase-I/II clinical trials.

Table 2 - bsAbs in early pipeline clinical trials with no results disclosed.

MOAs	Targets	Drug	Company	Phase (NCT/CTR)	Indication(s)
Simultaneous blockade of two signaling pathways to inhibit tumor growth
	EGFR × c-MET	EMB-01	Epimab	Phase I/II (NCT03797391)	Solid tumors
		MCLA-129	Betta Pharm.	Phase I/II (NCT04868877)	NSCLC
				Phase I/II (NCT04930432)	Solid tumors
Bridging immune cells with tumor cells for redirected cytotoxicity
	EGFR × CD3	BC3448	Wuxi Biocity	Phase I (ChiCTR2100053341)	Solid tumors
	DLL3 × CD3	BI 764532	Boehringer Ingelheim	Phase I (NCT04429087)	SCLC and other neoplasms
	HER2 × PD-1	SSGJ-705	Sunshine Guojian Pharm.	Phase I (NCT05145179)	HER2-expressing solid tumors
Targeting dual immunomodulatory molecules to promote immune responses
	PD-1 × CTLA-4	QL-1706	Qilu Pharm.	Phase Ib (NCT05171790)	Solid tumors
		QL-1706 + Chemo		Phase II (NCT05329025)	NSCLC
		SI-B003	Sichuan Baili Pharm.	Phase I (NCT04606472)	Solid tumors
	PD-1 × LAG3	EMB-02	EpimAb	Phase I/II (NCT04618393)	Solid tumors
		RO7247669	Roche	Phase I/II (NCT04140500)	Solid tumors
	PD-L1 × LAG3	IBI323	Innovent Biologics	Phase I (NCT04916119)	Advanced malignancies
	PD-L1 × TIM3	RO7121661	Roche	Phase I (NCT03708328)	Solid tumors
	PD-L1 × CD47	SG12473	Sumgen Biotech	Phase I (CTR20211029)	Malignant tumors
	PD-L1 × TIGIT	PM1022	BIOTHEUS	Phase I (CTR20220576)	Malignant tumors
		HLX301	Shanghai Henlius Biotech	Phase I/II (NCT05102214)	Solid tumors
	PD-1 × TIGIT	ZG005	Suzhou Zelgen Biopharma	Phase I/II (CTR20220021)	Solid tumors
		IBI321	Innovent Biologics	Phase I (NCT04911894)	Solid tumors
	PD-1 × TGF-β	JS-201	Shanghai Junshi Bioscience	Phase I (NCT04956926)	Malignant tumors
		JS-201 + Lenvatinib		Phase II (NCT04951947)	SCLC
	PD-L1 × 4-1BB	ES101	Elpiscience Biopharma	Phase I/II (NCT04841538)	Malignant thoracic tumors
		PM1003	BIOTHEUS	Phase I/II (ChiCTR2100052887)	Solid tumors
		QLF31907	Qilu Pharm.	Phase I (NCT05150405)	Malignant tumors
		LBL-024	Nanjing Leads Biolabs	Phase I/II (NCT05170958)	Solid tumors
		ATG-101	Antengene Biologics Limited	Phase I (NCT04986865)	Solid tumors

bsAbs: Bispecific antibodies; Chemo: Chemotherapy; c-MET: c-mesenchymal-epithelial transition factor; CTLA-4: Cytotoxic T lymphocyte-associated antigen-4; CTR: Clinical Trial Registry; DLL3: Delta-like protein 3; EGFR: Epidermal growth factor receptor; HER2: Human epidermal growth factor receptor-2; LAG3: Lymphocyte activation gene-3; MOAs: Mechanisms of action; NCT: National Clinical Trial; NSCLC: Non-small-cell lung cancer; PD-1: Programed cell death protein 1; PD-L1: Programed death-ligand 1; Pharm.: Pharmaceutical; SCLC: Small cell lung cancer; TGF-β: Transforming growth factor-beta; TIGIT: T cell immunoreceptor with Ig and ITIM domains; TIM3: T cell immunoglobulin and mucin-domain containing-3.

Several novel bsAbs simultaneously targeting two immune checkpoints are at the forefront of cancer clinical trials. These compounds include PD-L1 × CTLA-4 (QL-1706, SI-B003), PD-1 × LAG3 (EMB-02, RO7247669), PD-L1 × LAG3 (IBI323), PD-L1 × CD47 (SG12473), PD-L1 × T cell immunoreceptor with Ig and ITIM domains (TIGIT) (PM1022, HLX301), and PD-1 × TIGIT (ZG005, IBI321). The results from a pre-clinical trial of AK129 (an anti-PD-1 × LAG3 bsAb), reported at AACR 2022, are encouraging. It not only has excellent antigen binding properties to effectively block the PD-1/LAG3 signaling pathway, but also promotes the production of interleukin-2 (IL-2) and IFN-γ in peripheral blood mononuclear cells. In animal experiments, it has shown superior antitumor activity to relatlimab. These bifunctional agents have broad application prospects in the treatment of lung cancer. bsAbs that combine immune checkpoint blockade and stimulation of T-cell-costimulatory molecules are still at the early stages of clinical development. Phase-I/II trials are in progress for agents targeting PD-L1 × 4-1BB (ES101, PM1003, QLF31907, LBL-024, and ATG-101).

BiTEs are also attractive for bsAb development. BC3448 (anti-EGFR × CD3) demonstrates effective antitumor activities in preclinical studies. A phase I study of BC3448 in locally advanced and metastatic solid tumors is being conducted in China and finished the first patient dosing in January 2022. BI 764532 is an anti-DLL3 × CD3 bsAb. It is now in a phase-I study in patients with SCLC or other neuroendocrine neoplasms expressing DLL3.^[80] Differently, SSGJ-705 is a bsAb with one arm binding to the tumor cell surface antigen-HER2 and the other to PD-1, thereby forming a bridge mechanism as well as activating immune cells via PD-1 blockade. A phase-I clinical trial is currently evaluating the safety and efficacy of SSGJ-705 in patients with advanced or metastatic HER2-expressing solid tumors.^[81]

Importantly, multispecific antibodies are also in their early development stage. Simultaneous targeting of ≥3 TAA and immunomodulatory molecules might attain a higher efficacy than mono- or bi-specific antibodies. Numerous multispecific antibodies have shown effective preclinical outcomes. For example, HPN328, a trispecific T-cell-activating antibody targeting CD3, albumin, and DLL3 for treatment of SCLC has shown promising initial results, with three of nine SCLC patients showing tumor decrease >30%, as reported at ASCO 2022.^[82] It is also in a phase-I/II trial (NCT04471727) as monotherapy in advanced cancers associated with the expression of DLL3.^[82] An EGFR/c-MET/c-MET trispecific antibody, GB263T, is in a phase-I/II trial (NCT05332574) in advanced NSCLC and other solid tumors. NM21-1480 targeting PD-L1/4-1BB/human serum albumin (HSA) showed potent antitumor activity in preclinical studies and is on phase I/II clinical trial (NCT04442126) in advanced solid tumors.^[83] Despite the advances in the field of multispecific antibodies, there are multiple problems in the aspects of design, production, and clinical applications.^[84] Whether multispecific antibodies will lead to a breakthrough in lung cancer warrants further investigation.

Challenges and Perspectives

To date, several phase-I/II trials of bsAbs have shown potent antitumor efficacy, portraying a bright future for the use of bsAbs in the treatment of lung cancer. Nonetheless, the therapeutic application of bsAbs in lung cancer faces numerous challenges, as we list in the following part.

Insufficient tumor penetration of bsAbs

Solid tumors with high interstitial pressure and disorganized vasculatures form a natural physical barrier that limits the tumor penetration of bsAbs as well as infiltration of T cells into tumors.^[85] BsAbs with small molecular weights, such as PM8001, are superior to large bsAbs in tumor penetration. Combinatorial strategies with other immunomodulatory agents and developing new vehicles to deliver bsAbs to solid tumors are potential solutions to tackle this challenge. In addition, bsAbs that can cross the blood-brain barrier are also needed to target the central nervous system.

Selection of targets

Selecting appropriate targets in solid tumors is also challenging. Most bsAb targets under development are TAAs that are also expressed in normal cells. This issue narrows the therapeutic window of bsAbs. TSAs represent ideal targets for cancer therapy due to that targeting TSAs can enhance anti-tumor effect as well as reduce off-target toxicity compared with TAAs. However, additional high-quality “omics” data through sequencing technology and software algorithms are needed to determine the ideal targets. Moreover, multispecific antibodies that concurrently target multiple antigens on the surface of cancer cells may circumvent the off-target problem of bsAbs.

Adverse effects

Immunotherapeutic agents, such as biTEs, have inherent and potentially life-threatening adverse effects, especially CRS. Pretreatment with a corticosteroid is currently the standard treatment for CRS control. Whether corticosteroids might inhibit the anti-tumor efficacy of bsAbs needs further investigation. Using anti-cytokine antibodies and modifying bsAbs by changing or deleting the Fc region are feasible measures to reduce CRS.^[86] In addition, the influence of immunogenicity on the efficacy and safety of bsAbs cannot be ignored. Immunogenicity assessment as early as possible before clinical studies and development of more comprehensive detection methods may help overcome immunogenicity.^[87]

Biomarkers

Biomarkers that can predict the clinical response to bsAbs are still largely unknown, and only a few have been reported. The scarce achievement in this aspect may result from the specificity of dual targets, which hinders the building of pharmacokinetics/pharmacodynamics models.^[88] The relatively few results from the clinical trials of bsAbs may also increase the difficulty of biomarker tracing. Thus, additional clinical data are needed to predict relevant biomarkers.

Design of clinical trials

The complex structure and unique MOAs of bsAbs hamper designing risk controls and optimal dosing regimens in clinical trials. It is particularly important to scientifically set the initial drug dose and the gradient and speed of dose escalation. Establishing favorable animal model for preclinical evaluation is helpful for the design of phase I trials. The optimal dosing strategy for bsAbs should be comprehensively considered in combination with pharmacological, toxicological, pharmacokinetic, and pharmacodynamic data of drugs with the same target. Selecting ≥2 candidate dosing regimens within the safe dose range in extended studies might help determine the optimal dosing strategy.

Conclusion

With the recent advances in biotechnology and target identification, bsAbs have emerged as promising therapeutics against lung cancer. To date, one bsAbs drug has been approved by the FDA for the later-line setting of advanced lung cancer. More importantly, various preclinical trials and phase-I clinical studies of bsAbs in solid tumors are undergoing, and some of these bsAbs have been evidenced to have anti-tumor effects. Further investigation in target selection, molecular structure optimization, optimal dosing regimens, biomarker identification, and combinatorial therapies may help to improve the potency of bsAbs.

Taken together, the development of bsAbs has achieved dramatic progress in the field of lung cancer; the undergoing clinical trials of bsAbs will improve our understanding of the efficacy and safety of using bsAbs in the treatment of lung cancer. Ultimately, the clinical utilization of bsAbs might widen the landscape of treatment and serve as an important arsenal for patients with lung cancer.

Funding

This work was sponsored by grants from the National Natural Science Foundation of China (Nos. 82172869 and 81972167), the Program of Shanghai Academic Research Leader (No. 21XD1423200), and the program of Shanghai Shenkang Hospital Development Center (No. SHDC12019133).

Conflicts of interest

None.