Bispecific antibodies: a novel approach for targeting prominent biomarkers

ABSTRACT

Many types of cancers are prevalent in India and worldwide. Monoclonal antibodies (MAbs) are one of the major types of cancer therapeutics, which have included MAbs of hybridoma, chimeric, humanized, or human origin. MAbs are mostly generated currently by direct cloning from B cells. Bispecific antibodies (BAbs), as the name suggests, have two different antigen-binding domains in a single molecule and thus have dual functionality/specificity combined in a single antibody. In addition to the detection of two different antigenic molecules, the dual functionality of BAbs can be utilized to mount T-cell-mediated killing of tumor cells wherein one Fv binds to the tumor-specific antigen and the another recruits T cells to the site of action. Breast cancer and prostate cancer are among the most prevalent cancers in women and men, respectively. Biomarkers such as HER2 and ER/PR are expressed in breast cancer, while overexpression of hepsin and prostate-specific membrane antigen is observed in prostate cancer. Developing BAbs against these biomarkers may be a potent therapeutic option to target breast and prostate cancer, respectively. Therefore, an efficient method using recombinant DNA technology and mammalian cell culture platform is required to generate BAbs against specific diseases as biomarkers as well as for the generation of antibody-based therapeutics.

KEYWORDS:

• Bispecific antibodies
• biomarker
• cancer
• BiTE

Introduction

Monoclonal antibodies (MAbs) have revolutionized cancer treatment. Over the last few decades, they have evolved as significant contributors in cancer therapeutics that have been effective in addressing various oncogenic malignancies. More than 70 licensed MAbs have reached the market and are being widely used in treatment and diagnosis. According to Cancer Research UK, the global market for MAb drugs stood at 102 USD billion in 2018, and it is expected to grow at a CAGR of 8.5% to 2025.¹

With their high specificity and sensitivity, MAbs have emerged as a successful treatment option for cancers. MAbs work by killing the tumor cells either by direct action through activation of different signaling pathways such as Vascular Endothelial Growth Factor (VEGF) signaling, Endothelial Growth Factor-Related (EGFR) signaling, etc., or by the indirect pathway that involves stimulating the immune system components to act on the tumor cells. In another mode of action, MAbs work by vascular and stromal ablation of tumors.² Various mechanisms of action of MAbs are listed in Table 1.

Table 1. Mechanisms of action of MAbs (Adapted from Scott et al.)²

Direct tumor cell killing

●Cell surface receptor agonist activity (leading to programmed cell death)

●Cell surface receptor antagonist activity (inhibits signaling that reduces cell proliferation)

●Neutralization of cell surface by enzymes (leading to inhibition of different signaling pathways)

Immune-mediated tumor cell killing

●Complement system activation

●Induction of phagocytosis

●ADCC (antibody-dependent cell cytotoxicity)

●T-cell and B-lymphocyte activation

Vascular and stromal ablation

●Inhibition of stroma

●Compromising tumor angiogenesis

●Conjugated antibody for drug delivery at target site

MAbs have proven to be highly potent, specific, and relatively safe therapeutics for specific killing of tumor cells as opposed to conventional radiotherapy and chemotherapy, which are ‘systemic’ in treatment and produce various side effects in the patient. MAbs have certain shortcomings in terms of relatively high cost and short-lived response. Generating MAbs has been a slow process, although direct cloning from B cells has accelerated the discovery process. Much research is being applied toward devising efficient options for the diagnosis and treatment of cancer that can be used in combination with MAbs for better prognosis.

Cancer as a multi-factorial disease is not always treated as effectively as possible by single-target immunotherapy under various circumstances.³ Furthermore, the ability of cancer cells to constantly mutate can lead to resistance or lack of responsiveness to targeted therapeutics.⁴ Bispecific antibodies (BAbs) represent the next line of potential therapeutics that can be used in combination with the existing treatment options for better cancer prognosis and cure.

The concept of BAbs that do not occur in nature has its roots from the study in which Staerz et al. ⁵ demonstrated cancer cell lysis by engaging T cells.⁵ It was only after 25 y that research on BAbs was boosted when the first BAb, Blinatumomab/MT103 (bispecific for CD3 and CD19), was being tested in clinical trials.^6,7 This BAb, which is a bispecific T-cell engager (BiTE) in its structure,⁸ has been approved for acute lymphoblastic leukemia (ALL). BAbs have a dual functionality combined in a single antibody⁹ as depicted in Figure 1.

Figure 1. Basic architecture of a bispecific antibody. BAbs have two arms complementary to different antigens, which imparts dual specificity to the molecule. MAbs have both Fab fragments with the same specificity; i.e., they bind to a single antigen. The dual specificity enables BAbs to affect cell-specific drug delivery, cytotoxic T-cell interactions with tumor cells, and other immune-mediated actions on foreign antigens. MAbs are superior for opsonization or complement system activation-mediated clearance of antigens

BAbs are of two kinds: immunoglobulin G (IgG)-like and non-IgG-like molecules¹⁰ as depicted in Figure 2. IgG-like BAbs are capable of effector functions such as complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis, whereas non-IgG-like BAbs are small and can achieve deeper tissue penetration.¹¹ The IgG-like BAbs resemble natural IgG as characterized by the presence of three arms/binding sites: two antigen-binding Fab arms and an Fc arm. Some of the standard IgG-like BAb formats include Triomab, Immunotoxin, Knob-in-hole BAbs (KIH BAbs), and TriFabs. Triomabs have two Fabs binding to two different antigens at the same time and an Fc region enhanced to bind to immune cells to elicit ADCC and facilitate antigen presentation.¹² Immunotoxins have the Fc region tagged with toxins such as diphtheria toxin and are used for site-specific drug delivery and killing of target cells.¹³ KIH BAbs carry certain amino acid modifications at their Fc region that facilitate better heterodimerization and overcomes the mispairing problem that arises during assembly of antibody chains ex vivo.¹⁴ TriFabs have three Fab fragments, two for one antigen and the remaining one for other.¹⁵ Apart from these IgG-like BAbs, many such additional molecules are being developed, each with their respective function and utility.¹⁶

Figure 2. Different BAbs formats: (a) TrioMab; (b) immunotoxin; (c) KIH BAbs; (d) TriFabs; (e) BiTE; (f) bispecific nanobody; (g) diabody. (a) BiTe consists of a variable heavy and light domain joined by a linker. (b) Immunotoxin, wherein an antibody is linked to a toxin. (c) TrioMab is trifunctional, corresponding to three different targets at the same time. (d) TriFabs, BAbs having dual specificity with two regular Fab arms fused to an asymmetric third Fab module via linker peptides. (e) Bispecific nanobody. (f) Knob into hole bispecific antibody. (g) Knob into hole bispecific antibody with a common light chain

The non-IgG-like BAbs lack the Fc arm and possess only two antigen-binding Fab arms.¹⁷ Common non-IgG-like BAbs include BiTE, bispecific nanobody, and diabody. BiTE works by engaging the T-cells of the immune system at the site of infection.¹⁸ Bispecific nanobody is a compact molecule containing the variable heavy chains for the two antigens linked by a short peptide called the linker.¹⁹ Diabody is a relatively large molecule compared to a nanobody and contains the Fab fragments for the two antigens linked together.^20,21 Non-IgG-like BAbs have better tissue penetration, while IgG-like BAbs are more stable and easier to purify in downstream processing.

One of the essential goals of cancer immunotherapy is to redirect immune effector cells toward tumor cells, and BAbs have been designed to achieve this phenomenon.^22,23 A more focused approach toward utilizing BAbs in onco-immunotherapy is via the generation of BiTE. BiTE works by engaging T cells at the site of infection. They are responsible for the redirection of cytotoxic T cells against pathogenic target cells. CD3 mediates T-cell activation, and binding of anti-CD3 antibody can mimic the specific antigen recognition by T cells. BAbs capable of recognizing CD3 on the T cell and a second antigen on the surface of a tumor cell are being developed.²⁴ At present, over two dozen BAbs are in clinical development, with BAbs for EpCAM × CD3 and HER2 × CD3 being the most widely studied BAbs.²⁵ Both of these BAbs work on the principle of T cell recruitment and Fc-mediated effector function for EpCAM-positive tumor and Her2/Neu-positive advanced solid tumors, respectively.^26,27 Thus, the engagement of T cells for the eradication of tumors using BAbs is presently one of the most compelling concepts for the treatment of cancer. This methodology involves the identification of the biomarkers overexpressed under various cancers and developing antibodies that identify the same.

Apart from engaging T cells to the site of tumor cells, BAbs are being developed for varied applications such as (a) blocking of signaling pathways: HER2/HER3 signaling, IGF-1R/HER3 signaling, and EGFR/HER3 signaling, thus simultaneously neutralizing two targets with a single molecule;^28–30 (b) targeting tumor angiogenesis: RG7221 BAb targeting VEGFA and angiopoietin-2 (Ang-2) inhibited angiogenesis and tumor growth strongly as compared to single-pathway inhibitors;³¹ (c) BAbs have been designed for diagnostic assays to accurately detect bacterial or viral antigens, instead of antibodies generated by immune response, resulting in early-stage diagnosis and treatment of pathogens.³² Table 2 provides a list of clinically approved BAbs and BAbs under development.

Table 2. List of clinically approved and under development BAbs (https://clinicaltrials.gov)

BAbs	Format	Targets	Biological functions	Clinical Trial X Identifier	Diseases
Catumaxomab	Triomab	EpCAM × CD3	T-cell recruitment, Fc-mediated effector function	Approved in EU	EpCAM-positive tumor, malignant ascites
Ertumaxomab	Triomab	HER2 × CD3	T-cell recruitment, Fc-mediated effector function	Phase I/II, NCT01569412	Her2/Neu-positive advanced solid tumors
Solitomab (MT110, AMG 110)	BiTE	CD3 × EpCAM	T-cell recruitment	Completed phase I, NCT00635596	Solid tumors
BAY2010112	BiTE	CD3 × PSMA	T-cell recruitment	Phase I, NCT01723475	Prostatic neoplasms
rM28	Tandem scFv	CD28 × HMV-MAA	Retargeting autologous lymphocytes to tumor	Phase I/II, NCT00204594	Malignant melanoma
Indium-labeled IMP-205xm734	Unclear	CEA × in-labeled Peptide	Nuclear imaging	Phase I, NCT0018508	Colorectal cancer
LY3164530	OrthoFab-IgG	MET × EGFR	Blockade of two receptors	Phase I, NCT02221882	Neoplasms; neoplasm metastasis
IMCgp100	ImmTAC	CD3 × gp100	T-cell recruitment	Phase I, NCT01211262	Malignant melanoma
DT2219ARL	2 scFv linked to diphtheria toxin	CD19 × CD22	Targeting of protein toxin to tumor	Phase I, NCT00889408	Leukemia; lymphoma
Anti-CEAxanti-DTPA	scFv–IgG	CEA × di-DTPA-131I	Radioimmunotherapy	Complete phase II, NCT00467506	Medullary thyroid carcinoma
REGN1979	Unclear	CD20 × CD3	T-cell recruitment	Phase I, NCT02290951	CD20 + B cell malignancies
COVA322	IgG-fynomer	TNF-α × IL17A	Blockade of two proinflammatory cytokines	Phase I/II, NCT02243787	Plaque psoriasis
RG7221 (RO5520985)	CrossMAb	Ang-2 × VEGF	Two-ligand inactivation	Phase II, NCT01688206	Neoplasms
RG7716	CrossMAb	VEGF × Ang-2	Two-ligand inactivation	Phase II, NCT02484690	Wet AMD
MM-111	HSA body	HER2 × HER3	Blockade of two receptors	Completed phase I, NCT01097460	Breast neoplasms
RG7813 (RO6895882)	ScFv–IgG	CEA × IL2	Delivery of cytokines	Phase I, NCT02004106	Advanced and/or metastatic solid CEA+ tumors
RG7802	CrossMab	CEA × CD3	T-cell recruitment	Phase I, NCT02324257	Solid cancers
Blinatumomab	BiTE	CD3 × CD19	T-cell recruitment	Approved in USA	ALL
AMG 330	BiTE	CD33 × CD3	T-cell recruitment	Phase I, NCT02520427	Relapsed/refractory AML
MT111 (MEDI-565)	BiTE	CEA × CD3	T-cell recruitment	Completed phase I, NCT01284231	Gastrointestinal adenocarcinomas
FBTA05	TrioMab	CD20 × CD3	T-cell recruitment	Phase I/II, NCT01138579	Leukemia
TF2	Dock and lock	CEA × HSG	Radioimmunotherapy	Phase I, NCT00895323	Colorectal cancer
EGFRBi-aATC	T cells preloaded with BAbs	CD3 × EGFR	Autologous activated T cells to EGFR-positive tumor	Phase I/II, NCT02521090	Adult brain glioblastoma; adult gliosarcoma; recurrent brain neoplasm
MGD006	DART	CD123 × CD3	Retargeting of T cells to tumor	Phase I, NCT02152956	Relapsed/refractory AML
OMP-305B83	DVD-Ig	DLL4 × VEGF	Two-ligand inactivation	Phase I, NCT02298387	Advanced solid tumor malignancies
MGD007	DART	gpA33 × CD3	Retargeting of T cells to tumor	Phase I, NCT02248805	Colorectal carcinoma
GD2Bi-aATC	T cells preloaded with BAbs	CD3 × GD2	Activated T cells	Phase I/II, NCT02173093	Children and young adults with neuroblastoma and osteosarcoma
HER2Bi-aATC	T cells preloaded with BAbs	CD3 × HER2	Activated T cells	Phase I, NCT02470559	Ovarian, fallopian tube, or primary peritoneal cancer
MT112 (BAY2010112)	BiTE	PSMA × CD3	T-cell recruitment	Phase I, NCT01723475	Prostatic neoplasms
MEDI-565	BiTE	CEA × CD3	T-cell recruitment	Completed phase I, NCT01284231	Gastrointestinal adenocarcinomas
MDX447	2 (Fab’) was crosslinked	CD64 × EGFR	Active monocytes to kill tumor	Completed phase I, NCT00005813	Brain and central nervous system rumors
MM-141	scFv–IgG	IGF-IR × HER3	Blockade of two receptors	Phase I, NCT01733004	Hepatocellular carcinoma
MOR209/ES414	scFv–IgG	PSMA × CD3	T-cell recruitment	Phase I, NCT02262910	Prostate cancer
TargomiRs	Unclear	EGFR × EDV	Delivery of nanoparticles	Phase I, NCT02369198	Recurrent MPM and NSCLC
MSB0010841	Nanobody	IL-17A/F	Blockade of two proinflammatory cytokines	Phase I, NCT02156466	Psoriasis
Ozoralizumab (ATN-103)	Nanobody	TNF × HSA	Blockade of proinflammatory cytokine binds to HSA to increase half-life	Completed phase II, NCT01063803	Rheumatoid arthritis
SAR156597	scFv–IgG	IL4 × IL13	Blockade of proinflammatory cytokines	Completed phase I/II, NCT01529853	Idiopathic pulmonary fibrosis
AFM11	TandAb	CD30 × CD19	Redirecting of T cells	Phase I, NCT02106091	Relapsed and/or refractory CD19-positive B cell NHL
ALX-0061	Nanobody	IL-6 R × HSA	Blockade of proinflammatory cytokine, binds HSA to increase half-life	Completed phase I/II, NCT01284569	Rheumatoid arthritis
AFM13	TandAb	CD30 × CD16A	Active NK cells	Phase II, NCT02321592	Relapsed or refractory Hodgkin lymphoma

This review provides an insight into the conventional biomarkers expressed in the two most prevalent cancers in male and female, i.e., prostate and breast cancer, respectively, as mentioned in Table 3.

Table 3. Biomarkers expressed in the two most prevalent cancers in male and female, respectively

Gender (sex)	Prominent biomarker	References
Female	Breast cancer
	Her-2	33,34,35,36
	ER (estrogen receptor)	37,38,39
	PR (progesterone receptor)	40,41,42
Male	prostate cancer
	PSMA (prostate-specific membrane antigen)	43,44,45,46,47
	Hepsin	48,49,50,51

Common biomarkers in breast cancer

Breast cancer affects women in the middle and old age groups. According to WHO, breast cancer is among the leading causes of female deaths worldwide, with about 2.1 million deaths reported in 2018.⁵² The various biomarkers that are prevalent in breast carcinoma can be studied for the development of anti-marker sequences.

HER2

HER2 is a transmembrane protein receptor which belongs to Human Epidermal Growth Receptor Family and is a tyrosine kinase.³³ The HER family consists of four cell surface receptors (namely HER1, HER2, HER3, and HER4). HER2 acts in a phosphorylation pathway that transmits signals through site-specific phosphorylation and dephosphorylation of various domains of the HER2 receptor,³⁴ as depicted in Figure 3. Overexpression of the HER2 oncogene is likely to be both a prognostic and a predictive factor in patients with breast cancer.³³ The cell surface receptor exists as a homo- or heterodimer and facilitates growth-related signaling of cells. Overexpression on the surface results in enhanced responsiveness to growth factors and malignant growth.³⁵

Figure 3. Activation of various pathways involving HER family of receptors

Adapted from Ferreira et al.³⁵

According to the National Cancer Institute (NCI), overexpression of HER2 is the causal agent for breast cancer in about 30% of the patients. Furthermore, HER2 is a potential biomarker related to metastasis, relapse, and decrease in overall survival of the patient. The most recent work done on HER2 included the development of MAbs that attach to the receptor and block its function, thereby preventing malignant growth of tumors. Mainly, two MAbs by the name Transtuzumab (commonly called Herceptin) and Pertuzumab (widely called, Perjeta) are approved.³⁶ The development of BAbs against HER2 can be beneficial since:

1. HER2 overexpression has the most lethal effect on patients triggering metastasis, relapse, and lower immunity.

2. It has a high expression rate in ER-positive, PR-positive, and even node-negative breast cancer patients, apart from getting overexpressed in HER2-positive individuals.

3. Developing a technology as BAbs require a considerable amount of background information about the signaling of the biomarker, its protein structure, and properties. With great effort being put to unveil HER2 dynamics, developing BAbs on the same makes it a potential target molecule, well backed by available data and statistics.

ER/PR

ER/PR represent the progesterone receptor (PR) and estrogen receptor (ER) and are used in the evaluation of breast cancer.

Estrogen receptor

ER is a hormone-dependent receptor that binds to estrogen. It has been one of the most widely studied drug targets in devising treatment methods against breast cancer. It exists as a sequestered multi-protein inhibitory complex in the nucleus, by heat shock proteins (HSP, mainly 50, 70, and 90). Upon interaction with estrogen, the ER undergoes a conformational change and becomes a dimer. This dimerized form then interacts with various estrogen receptor elements (ERE) to control the transcription of several genes. Apart from affecting the genomic mechanisms, ERs also exist in membrane form and bring about rapid, direct action of estrogen on to the cell. They bring about changes in membrane potential by modulating sodium and calcium ion transport and activating nitric oxide pathway and mitogen-activated protein kinase (MAPK) pathway. Two significant classes of ERs are present in a typical mammary cell, namely ERα and ERβ; both of these mediate gene expression with differences in their mode of action:

1. Complexed ERβ is a weak activator than ERα.

2. While ERα is mostly involved in cell proliferation, ERβ is linked to suppression of proliferation.

3. In some instances, ERβ works by modulating/attenuating the action caused by ERα.

Under normal conditions, a breast cell expresses an almost negligible amount of ER, which stimulates cell proliferation. ER is often overexpressed in breast cancer. Hence, during the progression of breast cancer, the production of ERα rises exponentially. The production of ERβ decreases rapidly, given its function of limiting cell growth and division.³⁷ Breast cancer caused by the overexpression of estrogen receptors is regarded as ER-positive breast cancer. The few causes of an increase in expression of ER can be:

1. Overproduction of estrogen in the body or administration of estrogen through oral contraceptives or hormone replacement therapy.

2. Binding of mutated forms of estrogen to the preexisting ERs can cause a rapid increase in the production of new, unlinked estrogen receptor molecules.³⁸

According to the American Cancer Society, two out of three breast cancers are hormone dependent and ER positive.³⁹ Furthermore, apart from being overexpressed in the case of ER-positive cancers, ER is a common biomarker showing enhanced production in other types of breast cancer as well as HER2-positive and PR-positive cancers.

Progesterone receptor

PR is also a hormone-dependent receptor that binds to progesterone, commonly referred to as the pregnancy hormone. Under normal conditions, a mammary cell lacks any PR. PR is expressed in the self-altered cancer cell surface and cytosol. PR functions as a ligand-activated transcription factor molecule acting on both genomic and non-genomic levels. The common isoforms PR-A and PR-B are produced by different post-transcriptional modification of the same gene product. PR-B is a full-length receptor, while PR-A lacks the N-terminal 164 amino acids. Both PR-A and PR-B regulate the same set of target genes. PR-C is another isoform, found in the reproductive tissues, and involved in the induction of labor at the time of pregnancy. At the non-genomic level, the PRs work by activating various signal transduction pathways, many of which are involved in pro-proliferative signaling in the breast as depicted in Figure 4.

Figure 4. Growth factor-induced signaling by progesterone receptor isoforms

Adapted from Daniel et al. ³⁴

About two-thirds of ER-positive breast cancers are also PR positive and vice-versa.⁴¹ Available technologies target ER and PR overexpression in breast cancer using hormone therapy, also known as endocrine therapy, as a mechanism to stop the growth of hormone-sensitive tumors. It involves administering specific drugs that block the ER and PR, thereby preventing any further hormone-induced signaling.⁴² It also inhibits uncontrolled cell proliferation and differentiation. However, hormone therapy has been found to have mild-to-severe side effects such as nausea, fatigue, blood clots in brain, bone and muscle changes, etc. It also renders the body weak, making it susceptible to secondary infections. Development of BAbs against ER/PR can prove beneficial as follows:

1. High incidence of double-positive hormone receptor in breast cancer cases. A BAb having dual targeting for ER and PR should be more effective than a MAb against either ER or PR.

2. Conserved nature of receptor sequences, which makes it relatively easy to design complementary sequences. A BAb having one arm targeting ER/PR because of the conserved sequences and another arm targeting CD3 will result in the recruitment of T-cells and subsequent activation of T-cells at the target site.

Common biomarkers in prostate cancer

Prostate cancer is the second most prevalent cancer affecting males after lung cancer. According to WHO, 1.3 million cases of prostate cancer were reported in 2018, accounted for ~360,000 deaths.⁵² Two biomarkers expressed during prostate cancer have been the target of BAb development.

PSMA

Prostate-specific membrane antigen (PSMA) is a cell membrane-bound glycoprotein uniquely expressed in large amounts in the vasculature of prostate tumor cells and mostly absent in the vasculature of healthy prostate cells. It consists of a short intracellular domain of 18 amino acids, a transmembrane domain of 24 amino acids, and a glycosylated extracellular domain of 706 amino acids.⁴³ PSMA has two unique enzymatic functions:

1. Folate hydrolase activity: Cleaves terminal glutamates from folypoly-γ-glutamates to produce folates and their subsequent uptake.

2. NAALADase activity: Cleaves terminal glutamate from neuro-dipeptide, n-acetyl-aspartyl glutamate (stands for NAAG, it is a chemical that remains localized in neuronal synapse and produces neurotransmitter glutamates upon being metabolized)

Like any other membrane-bound receptor, PSMA also gets recycled through clathrin-coated pit,⁴³ and overexpression of PSMA is a critical feature of prostate cancer. The antigen plays an essential role in carcinogenesis and tumor angiogenesis. However, the mechanism of action of PSMA in cancer cells is unknown. A hypothesis regarding the role of PSMA in metastasis is that the antigen works by undergoing glycosylation, and the alteration in this process is responsible for carcinogenesis. Thus, due to its exciting yet unexplained distribution, it can serve as an essential biomarker for detecting prostate cancer.

PSMA is an excellent diagnostic target because of the following reasons:

1. It primarily expresses in prostate tumor cells with minimal expression in other tissues; hence, the site-specific action of the anti-PSMA antibody is assured.

2. Expression in prostate tumor cells is 1000-fold higher than in healthy prostate cells, making the cancer cells to be detected easily.

3. It is not released into circulation, thereby reducing the chances of side effects or allergy.⁴⁴

Current immunological interventions for diagnosis and therapeutics that depend on using PSMA target the heavy extracellular domain of the biomolecule as the J591 MAb developed with high binding affinity.⁴⁵ J591 is in Phase 1/2 study.⁴⁶ Capromab is another approved anti-PSMA antibody,⁴⁷ but since it targets the intracellular domain of PSMA, it is not useful on viable cells. While developing MAbs that target the extracellular domain can be advantageous, generating BAbs instead would also bring in the advantages of immune cell-assisted killing of the tumor cells.

HEPSIN

Hepsin is a type-2 transmembrane serine protease frequently overexpressed in prostate cancer cells. It is a 417 amino acid protein composed of a short N-terminal cytoplasmic domain, a transmembrane domain, and a single scavenger receptor cysteine-rich domain that packs tightly against the C-terminal protease domain.⁴⁸

Several studies have reported high expression levels of hepsin in late-stage prostate cancers. The exact role of hepsin in prostate cancer progression remains unclear. However, some recent insights have been provided by Zhang et al.,⁴⁹ who reported that hepsin might be acting on the translational machinery to suppress the expression of CDK11p58, a protein responsible for pro-apoptotic signaling in prostate cancer. There is also accumulating evidence that hepsin might be involved in promoting the invasive capacity of the tumor and even metastasis. Upregulation of hepsin in the prostate epithelium of mice is observed to cause disorganization of the basement membrane (BM) of the prostate in probasin (PB) promoter-driven hepsin transgenic mice.⁵⁰

Immunohistochemical staining using MAbs against hepsin developed in hepsin-knockout mice showed weak hepsin expression in tissues from healthy prostate, benign prostatic hyperplasia, or low grade (2/3) prostate cancer. In contrast, hepsin expression is elevated in advanced prostate tumors (grades 4/5) and bone metastasis, thereby confirming mRNA expression studies.⁵¹ Various MAbs against hepsin are being developed. One such antibody is Fab 25,⁵³ which follows noncompetitive inhibition kinetics while binding to hepsin. Thus, BAbs against hepsin can be highly efficient for prostate cancer.

Advantages of BAbs relative to MAbs

• (1) They are immunological molecules with dual functionality.

BAbs offer dual functionality since a single antibody molecule can interact with two surface antigens at the same time.

• (2) They offer a myriad of features.

BAbs have a highly specialized and wide range of functions such as recognizing two different antigens simultaneously, targeting various disease mediators dually, or delivering payloads to targeted sites.

• (3) They can target multiple diseases.

They are next-generation diagnostic options not only for various cancers but also for a myriad of other diseases including genetic disorders, bacterial diseases, and HIV infections.³²

• (4) They have significant advantages for cancer immunotherapy.

An added advantage is the ability to recognize tumor cells accurately and without using invasive techniques. BAbs allow for direct interaction of immune cells (NK cells, T-cells) with tumor cells. They also enhance the process of killing target cells by blocking more than one signaling pathway simultaneously.⁵⁴

• (5) BiTE is a type of BAb that allows for natural recruitment of T-cells on tumor cells.

BiTEs facilitate direct contact of tumor cells with host T cells, thereby enhancing T-cell potency for acting on tumor cells by creating suitable micro-environments for the release of cytotoxic mediators. BiTEs also can trigger serial killing of tumor cells by a single T cell.⁵⁴

• (6) Usage of BAbs for delivering payloads to target sites.

Given their dual specificity, BAbs can bind to cell surface antigens and molecular payloads simultaneously, thereby serving as excellent delivery systems for transporting cytotoxic entities to malignant cells.^55-57

Conclusion

BAbs are an important therapeutic in the field of onco-immunotherapy. Eliminating tumor growth is one of the most important mechanisms that is widely targeted for treating cancer. BAbs can be used in combinatorial therapy with current drugs and can be a cost-effective treatment for cancer. Apart from enhancing T-cell stimulation and tumor cell-binding properties of BAbs, other modifications such as improving the ADCC and Complement-Dependent Cellular Cytotoxicity (CDCC) activity of the molecule also can be explored. Furthermore, developing BAbs against biomarkers for breast and prostate cancer will be beneficial in detection and effective treatment. BAbs should add to the field of immunotherapy as a theragnostic against various cancers.

BAbs also have shortcomings.

1. The molecules pose difficulty in their downstream processing.

2. These molecules are mostly short-lived.

3. They may be immunogenic due to their non-human nature.

4. Heterodimerization of antibody chains from different sources can be problematic, thus reducing the efficiency of developing these drugs.

MAbs have evolved as site-specific treatments and will continue to have a durable position in the market and in the therapeutic armamentarium.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Abbreviations

BAbs	=	Bispecific antibodies
MAb	=	Monoclonal antibody
IgG	=	Immunoglobulin
BiTE	=	Bispecific T-cell engager
HER2	=	Human Epidermal Growth Receptor 2
ER/PR	=	Estrogen receptor/progesterone receptor
CD3	=	Cluster of differentiation 3
PSMA	=	Prostate-specific membrane antigen
Fc	=	Fragment crystallizable
Fab	=	Fragment antibody

Additional information

Funding

This work was supported by the Science and Engineering Research Board [ECR/2016/001752].