Preclinical evaluation of ISH0339, a tetravalent broadly neutralizing bispecific antibody against SARS-CoV-2 with long-term protection

Abstract

Background: Ending the global COVID-19 pandemic requires efficacious therapies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nevertheless, the emerging Omicron sublineages largely escaped the neutralization of current authorized monoclonal antibody therapies. Here we report a tetravalent bispecific antibody ISH0339, as a potential candidate for long-term and broad protection against COVID-19.

Methods: We report here the making of ISH0339, a novel tetravalent bispecific antibody composed of a pair of non-competing neutralizing antibodies that binds specifically to two different neutralizing epitopes of SARS-CoV-2 receptor-binding domain (RBD) and contains an engineered Fc region for prolonged antibody half-life. We describe the preclinical characterization of ISH0339 and discuss its potential as a novel agent for both prophylactic and therapeutic purposes against SARS-CoV-2 infection.

Results: ISH0339 bound to SARS-CoV-2 RBD specifically with high affinity and potently blocked the binding of RBD to the host receptor hACE2. ISH0339 demonstrated greater binding, blocking and neutralizing efficiency than its parental monoclonal antibodies, and retained neutralizing ability to all tested SARS-CoV-2 variants of concern. Single dosing of ISH0339 showed potent neutralizing activity for treatment via intravenous injection and for prophylaxis via nasal spray. Preclinical studies following single dosing of ISH0339 showed favorable pharmacokinetics and well-tolerated toxicology profile.

Conclusion: ISH0339 has demonstrated a favorable safety profile and potent anti-SARS-CoV-2 activities against all current variants of concern. Furthermore, prophylactic and therapeutic application of ISH0339 significantly reduced the viral titer in lungs. Investigational New Drug studies to evaluate the safety, tolerability and preliminary efficacy of ISH0339 for both prophylactic and therapeutic purposes against SARS-CoV-2 infection have been filed.

Statement of Significance: ISH0339 is a novel tetravalent broadly neutralizing bispecific antibody with long-term protection that has demonstrated excellent anti-SARS-CoV-2 activities against all widespread viral subtypes and favorable safety profile, with potential for both prophylactic and therapeutic purposes against emerging SARS-CoV-2 infection.

INTRODUCTION

Monoclonal antibodies (mAbs) are a promising class of therapeutics against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To date, multiple studies have reported the discovery and characterization of potent neutralizing mAbs, most of which target the receptor-binding domain (RBD) of the S protein of SARS-CoV-2 and block the binding between the S protein and the host receptor, human angiotensin-converting enzyme 2 (hACE2) [1–8]. There are also some neutralizing mAbs that target non-blocking epitopes of the RBD or N-terminal domain of the S protein of SARS-CoV-2 [9]. However, when selective pressure is applied in these anti-viral therapies, the emergence of escape mutants of virus is a major public concern [10–15].

Since its emergence in late 2021, the Omicron variant of SARS-CoV-2 has led to the emergence of numerous subvariants, including BA.1, BA.1.1, BA.2, BA.4, BA.5, BA.4.6, BF.7, BA.2.75, BQ.1, BQ.1.1 and XBB.1.5, that continue to evade vaccine-induced and infection-induced immunity [13, 16–18]. Mutation is a natural appearance that occurs during the replication cycle of all viruses and SARS-CoV-2 is no exception. However, viral mutations can lead to off-target effects of existing drugs and vaccines, thus allowing the virus to escape immunity [19, 20]. To protect against the mutational virus escape, cocktail strategies have been reported [16, 18, 19], but it increases manufacturing costs and volumes, indicating that it might not be the ideal strategy to meet the wide spectrum and long-term demand for SARS-CoV-2 therapeutics. Bispecific antibodies (bsAbs) take advantage of two diverse mAbs and can target two different antigen-binding epitopes with one molecule. Several studies showed that bsAbs exhibit enhanced breadth and potency than parental mAbs [21, 22]. Therefore, bsAb is an effective strategy in anti-infectious diseases drug development.

ISH0339 is a novel tetravalent broadly neutralizing bispecific antibody that binds specifically to two different epitopes of SARS-CoV-2 RBD and contains an engineered Fc region for prolonged antibody half-life. In this study, four structural-type bsAbs were designed based on these two parental mAbs, ISH0339-85 and ISH0339-151, which were characterized as potent non-competing neutralizing antibodies. The candidate ISH0339 was selected using ISH0339-151 as full-length IgG with two scFvs of ISH0339-85 fused to the N-terminal of light chains of ISH0339-151. ISH0339 showed excellent binding and neutralizing activities against emerging SARS-CoV-2 Delta and Omicron sublineages. In a mesocricetus auratus live virus challenged model, ISH0339 showed potent anti-SARS-CoV-2 activities in intravenous (IV) injection for treatment and nasal spray for prophylaxis. Furthermore, Fc engineering was introduced to ISH0339 for a prolonged half-life, which can delay the metabolism of the drug and provide long-term protection. Therefore, ISH0339 is a novel tetravalent broadly neutralizing bispecific antibody that has demonstrated excellent anti-SARS-CoV-2 efficacy and favorable safety profile, with potential for both prophylactic and therapeutic purposes against emerging SARS-CoV-2 infection.

MATERIALS AND METHODS

Binding of antibodies to RBDs by enzyme-linked immunosorbent assay

Test article ISH0339-85, ISH0339-151 and bsAbs was generated at SunHo (China) BioPharmaceutical Co., Ltd. 100 ng of SARS-CoV-2 RBD proteins from Wild type strain, variants Delta, BA.1, BA.1.1, BA.2, BA.4/BA.5, BA.4.6/BF.7 (ACRO Biosystems) were coated into 96-well plates for overnight at 4°C. The plates were blocked with blocking solution for 1 h. ISH0339–85, ISH0339–151, bsAbs and isotype control were diluted and applied to the enzyme-linked immunosorbent assay (ELISA) plate at 37°C for 1 h. After washing, secondary antibody was added for 45 min and then developed with Tetramethylbenzidine (TMB) peroxidase substrate and terminated with 1 M HCL. Absorbance at 450 nm (A450) was determined by Tecan sunrise (Tecan).

Epitope binning of ISH0339-85 and ISH0339-151

ISH0339-85 and ISH0339-151 were diluted to 2 μg/ml and coated into a 96-well microplate in 100 μl/well for 4°C overnight. The plates were blocked with blocking solution for 1 h. Competitor ISH0339-85 and ISH0339-151 were premixed with 0.5 μg/ml BA.1 RBD (ACRO Biosystems) at 100 μg/ml, respectively, and blank control was 3% non-fat milk powder with 0.5 μg/ml BA.1 RBD (ACRO Biosystems). The antibody premixed with RBD was added to the 96-well plate and incubated at 37°C for 45 min. After washing the plate, secondary antibody was added for 45 min. Followed by washing, the plate was then developed with TMB peroxidase substrate and terminated with 1 M HCL. Absorbance at 450 nm (A450) was determined by Tecan sunrise (Tecan).

Binding affinity of ISH0339 with FcRn by biofilm layer interferometry

The SA Biosensors (ForteBio. Inc.) were pre-wet with phosphate-buffered saline with Tween detergent (PBST) buffer for 20 min. Biotinylated Human FcRn/FCGRT &B2M Heterodimer Protein (ACRO Biosystems) diluted to 1 μg/ml with PBST was added to the SA Biosensors to reach 1.5 nm in thickness. ISH0339 and control antibody that did not contain the triple mutations M252Y/S254T/T256E (YTE) in the Fc region were diluted into 300 μg/ml using PBST, added to the above sensor, associated for 120 s, and dissociated for 120 s. The dissociation constant (KD) was calculated by fitting the binding dissociation curves of ISH0339 and control antibody bound to FcRn, respectively.

SARS-CoV-2 RBD/hACE2 blocking assay

Antibodies were diluted and incubated with SARS-CoV-2 BA.1, BA.2.75 and BQ.1.1 RBD proteins (ACRO Biosystems) for 30 min at room temperature. Mixed samples were added to HEK293-Hu ACE2 cells (ACRO Biosystems) and incubated for 1 h at room temperature. After washing the cells, the secondary antibody Anti-6X-his tag antibody-FITC (Abcam) was added at 1:500 and incubated for 30 min at room temperature, which were further analyzed by flow cytometry. Data were measured using ACEA NovoCyte (Agilent), and IC50 values were analyzed with GraphPad Prism 6.0 software.

SARS-CoV-2 pseudovirus neutralization assay

Antibodies were diluted and incubated with SARS-CoV-2 pseudovirus (ACRO Biosystems) for 1 h at room temperature. The HEK293-Hu ACE2 cells (ACRO Biosystems) were added to the culture plates at 5E4/well and cultured in the incubator for 48 h. After that, 100 μl of medium was discarded, and 100 μl of fluorescent enzyme chromogen was added immediately, and incubated for 5 min at room temperature. The chemical fluorescence signal was measured by Tecan sunrise (Tecan), and IC₅₀ values were analyzed with GraphPad Prism 6.0 software.

SARS-CoV-2 authentic virus neutralization assay

Vero E6 cells were seeded in 96-well plates prepared 1 day before use and grown to ⁓85% confluence in these assays. ISH0339 was diluted to a starting concentration of 500 ng/ml, then 2-fold gradient dilution with 10 gradients of triplicate wells per gradient, while doing a positive control (PC) well with only virus and a negative control (NC) well with only cells. An equal volume of 100 TCID50 BA.2 and BA.5 (The Kunming Institute of Zoology, Chinese Academy of Sciences) of virus was added to each diluted sample respectively, and incubated in a 37°C, 5% CO₂ incubator for 1 h, while preparing for both positive and NCs. The mixture of the above samples, the positive and NCs were, respectively, added to the cell plates coated with Vero-E6, followed by incubation in 37°C, 5% CO₂ for 4–6 days. Cytopathic effect (CPE) in each well was observed and recorded. The dilution ratio of the sample with ˃50% neutralization effect was calculated based on the cytopathic condition and was used as the neutralizing titer of the sample.

Prophylaxis and therapeutic in mesocricetus auratus live virus challenged model

Syrian hamsters were used for SARS-CoV-2 prophylaxis and therapeutic studies (The Kunming Institute of Zoology, Chinese Academy of Sciences). In each group, eight Syrian hamsters (mesocricetus auratus) per group were challenged with 1 × 10⁴ TCID₅₀ BA.2 (The Kunming Institute of Zoology, Chinese Academy of Sciences). At 2 h prior hamster virus challenge, ISH0339 was administered in nasal spray at a dose of 50 mg/kg in the prophylaxis group. In the therapeutic group, low-dose and high-dose of ISH0339 (25 mg/kg and 50 mg/kg) were injected singly into the hamsters in intraperitoneal administration at 2 h after hamster virus challenge. Hamsters were sacrificed and necropsied 3 days after challenge with virus. Hamsters were treated with PBS in the control group. The amount of virus ribonucleic acid copies per gram of tissue was determined using a RT-qPCR assay. The care or use of animals had been reviewed and approved by institutional animal care and use committee (IACUC).

Pharmacokinetic analysis of single-dose ISH0339 administration

In the single-dose study, 36 Sprague–Dawley rats [Medicilon Preclinical Research (Shanghai) LLC] were assigned to six groups. Animals were administered with single dose of 25, 50, 100 and 300 mg/kg ISH0339 by IV bolus administration or 26.5 and 53.0 mg/kg ISH0339 by intranasal administration on day 1. Blood samples for pharmacokinetic analysis were collected before the intervention and at 0.083, 1, 4, 8, 24, 48, 96, 144, 216, 312, 480, 648 and 816 h post administration. Phoenix WinNonlin 7.0 software (Certara) for non-atrioventricular model was used for calculation of pharmacokinetic parameters, which automatically selects at least three elimination phases before the end elimination for half-life calculation.

In vivo and ex vivo biodistribution study

ISH0339 was conjugated with Alexa Fluor 750 dye for in vivo imaging studies. Briefly, 11.235 mg/ml solution of ISH0339 in sodium bicarbonate buffer (pH 8.3) was reacted with Alexa Fluor 750 succinimidyl ester (AF750-NHS; Thermo Fisher Scientific) in the presence of dimethyl sulfoxide at a molar ratio of 1:10 (protein to fluorescent probe) at room temperature for 1 h. Unreacted dye was removed by desalter, and the labeled antibody concentration was 11 mg/ml, washed in 20 mM His/His-HCl, 200 mM Trehalose, 0.2 g/l Polysorbate80 (pH 6.0).

C57BL/6 N mice were anesthetized by inhalation of 2% isoflurane and placed in a supine position (Nanjing Clinbridge Biotech Co., Ltd). The mice were administered Alexa Fluor 750-labeled ISH0339 into nostrils using a pipet tip to achieve a final dose of 0, 1, 3 and 10 mg/kg body weight. The mice were then imaged at 2 min, 16 min, 2 h and 24 h in vivo after administration (fluorescence excitation wavelength 740 nm, emission wavelength 790 nm, auto-exposure setting, six mice/group) using an IVIS Lumina XRMS Imager (PerkinElmer). The remaining mice were euthanized at 2, 24, 48 and 96 h after administration, the heart, lungs, liver, spleen, kidneys, brain, jejunum, bladder and nasal cavities were excised and imaged (three mice per group). Regions of interest were drawn, and the radiant efficiency [(p/s/cm²/sr)/(μW/cm²)] was measured. All images were processed using Living Image software (PerkinElmer), and the same fluorescence threshold was applied for group comparison.

Extended toxicity study of single-dose ISH0339

A total of 180 Sprague–Dawley rats (90 per sex) were randomly assigned into six groups. In total, 15 female and 15 male rats received a single dose of 0, 50, 100 or 300 mg/kg ISH0339 by IV bolus or 30 and 100 mg/kg ISH0339 by intranasal administration [Medicilon Preclinical Research (Shanghai) LLC]. All animals (10 rats/sex/group) from dosing phase were terminated on day 2 after administration. All recovery animals (5 rats/sex/group) were necropsied at the completion of a 2-week recovery period after cessation of dosing (day 15) to assess the reversibility of any potential treatment-related effects.

Criteria for evaluation included viability (mortality and moribundity), clinical observations (including local tolerance at injection sites), body weights, food consumption, ophthalmology, body temperature, clinical pathology (hematology, serum chemistry, coagulation and urinalysis), immunophenotyping, gross necropsy, organ weights and histopathology.

Pharmacokinetic analysis of ISH0339

An indirect antigen ELISA assay was used for the detection of ISH0339 in rat serum (Medicilon Preclinical Research, Shanghai). The capture agent was SARS-CoV-2 S RBD，His Tag (ACRO, SPD-C522f), coated onto 96-well ELISA plates. After overnight incubation, the plates were blocked followed by the addition of the samples. After washing away any unbound substances, Peroxidase-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson, 109-035-098) was added to bind with ISH0339. Then the substrate was added and reacted with the peroxidase to create a colorimetric signal that was proportional to the amount of ISH0339.

RESULTS

Biological properties of two parental neutralizing mAbs ISH0339-85 and ISH0339-151

ISH0339 is a novel tetravalent broadly neutralizing bispecific antibody that binds specifically to two different epitopes of SARS-CoV-2 RBD protein and is composed of two non-competing parental mAbs, ISH0339-85 and ISH0339-151. ISH0339-85 and ISH0339-151 were both obtained from hybridoma campaigns. ISH0339-85 was generated by immunizing and screened with Omicron BA.1 RBD, while ISH0339-151 was derived from immunization with Delta RBD and cross screened with Omicron BA.1 RBD. To identify the biological properties of the two parental neutralizing mAbs, we performed binding, blocking and neutralization assays to ISH0339-85 and ISH0339-151, as we look for mAbs with distinct epitopes for bsAb generation that can exhibit broad neutralization activity and be resistant to potential viral escaping.

Binding activities of ISH0339-85 or ISH0339-151 to RBD proteins from various SARS-CoV-2 variants were analyzed by ELISA, indicating that ISH0339-85 could bind to SARS-CoV-2 RBD of Wild Type strain, variants Delta，BA.1 and BA.2，but not to BA.1.1, while ISH0339-151 could bind to all those strains (Fig. 1A and B). We further investigated the blocking activity of ISH0339-85 or ISH0339-151 to block the binding of BA.1 RBD to hACE2. The results revealed that ISH0339-85 could not block the binding of BA.1 RBD to hACE2, while ISH0339-151 possessed an excellent blocking activity (Fig. 1C). The above data indicated that ISH0339-85 and ISH0339-151 recognize distinct epitopes on RBD domain in non-competitive manner, which was further validated by epitope binning assay (Supplementary Table 1).

Figure 1.

Basic characteristics of two parental neutralizing mAbs of ISH0339. (A) ELISA binding capability of ISH0339-85 to SARS-CoV-2 RBD of Wild Type strain, Delta，BA.1，BA.1.1 and BA.2. (B) ELISA binding reactivity of ISH0339-151 to SARS-CoV-2 RBD of Wild Type strain, Delta，BA.1，BA.1.1 and BA.2. (C) ISH0339-85 and ISH0339-151 for blocking RBD and hACE2 binding. (D) The neutralizing activity of ISH0339-85 and ISH0339-151 against pseudovirus for BA.1.

Pseudovirus neutralization assay was used to detect the neutralizing activity of ISH0339-85 and ISH0339-151, demonstrating that both ISH0339-85 and ISH0339-151 could efficiently neutralize the BA.1 pseudovirus, with IC₅₀ values of 2.30 and 2.74 ng/ml, respectively (Fig. 1D). Taken together, these results indicated that ISH0339-85 and ISH0339-151 have potent neutralizing activity and possess different epitopes for ideal bsAbs generation. The humanization of ISH0339-85 and ISH0339-151 were obtained, which also showed similar biological activity as the chimeric parental antibodies (Supplementary Fig. 1).

Design and characterization of SARS-CoV-2-specific tetravalent bsAbs ISH0339, using ISH0339-85 and ISH0339-151

To obtain functional bsAb, we designed four IgG-scFv structural-type bsAbs (ISH0339-a, ISH0339-b, ISH0339-c and ISH0339-d) based on the above two neutralizing mAbs that targeted SARS-CoV-2 RBD different epitopes, ISH0339-85 and ISH0339-151 (Fig. 2A). Specifically, the ISH0339-85 scFv was fused to the C terminus and N terminus of the ISH0339-151 heavy chain by a GGGSGGGSGGGS linker in ISH0339-a and ISH0339-b, respectively. For ISH0339-c and ISH0339-d, the ISH0339-85 scFv was fused to the C and N terminus of the ISH0339-151 light chain by a GGGSGGGSGGGS linker respectively. Meanwhile, we designed Fc mutants of these structural-type bsAbs for prolonged half-life, named as ISH0339-a(YTE), ISH0339-b(YTE), ISH0339-c(YTE) and ISH0339-d(YTE). All these bsAbs folded correctly, and soluble proteins could be obtained. Binding activity of these bsAbs and ISH0339-151 to the BA.1 RBD proteins was analyzed by ELISA. The results revealed that all of these bsAbs had excellent binding affinity and most of them were better than ISH0339-151 (Fig. 2B). Among these, the binding affinity of the ISH0339-d structural format is higher than the other structural formats. Moreover, the orientation and distance of the four binding sites in ISH0339-d structural-type may favor the synergistic binding of two distinct epitopes on the same S protein and simultaneous neutralization of two S proteins by the tetravalency. Therefore, ISH0339-d(YTE) was selected as the final candidate molecule and named as ISH0339. Biofilm layer interferometry analysis showed that binding affinity of ISH0339 that contained a YTE mutation in the Fc region to FcRn was 9.7-fold higher than that of control wild type Fc, with KD values of 0.703 μM and 6.85 μM respectively (Supplementary Table 2)，indicating a prolonged half-life in vivo.

Figure 2.

Design and characterization of ISH0339 bsAbs. (A) Formats of the four structural-types ISH0339 bsAbs designed. The formats of ISH0339-a, ISH0339-b, ISH0339-c and ISH0339-d are IgG-scFv based with or without YTE Fc engineering. Antibody domains are colored according to their architecture (green, Constant region of ISH0339-151; blue, Variable region of ISH0339-151; yellow, scFv of ISH0339-85) (B) ELISA binding (OD450) reactivity of ISH0339-151, ISH0339-a, ISH0339-b, ISH0339-c and ISH0339-d to BA.1 RBD.

Biological characterization of ISH0339

To validate the biological function of tetravalent bispecific antibody ISH0339, we performed binding, blocking and neutralization assays with both pseudovirus and authentic virus. We first checked whether ISH0339 showed distinguished binding against the newly emerged SARS-CoV-2 variants, BA.4/BA.5 and BA.4.6/BF.7, compared with parental mAbs of ISH0339-85 and ISH0339-151. ELISA results showed that ISH0339 had much stronger binding affinity to BA.4/BA.5 RBD due to synergistic effects than both ISH0339-85 and ISH0339-151 alone, with EC50 values of 19.24 ng/ml, 86.98 and 157.2 ng/ml, respectively (Fig. 3A). More strikingly, ISH0339-85 completely lost the binding affinity for BA.4.6/BF.7, while ISH0339 still maintained high binding activity for this widespread virus mutant strain due to contribution of ISH0339-151 (Fig. 3B). The synergistic effect of ISH0339 was also reflected in the data on blocking assay which showed that ISH0339 had 1.8-fold higher blocking activity than ISH0339-151, while ISH0339-85 had no blocking activity as expected (Supplementary Fig. 2). To further confirm the broadly binding activity of ISH0339, we also examined the binding of ISH0339 for RBD proteins from different strains. Our results revealed that ISH0339 showed broad-spectrum binding activity to SARS-CoV-2 RBD proteins from Wild Type, Delta, BA.1, BA.1.1, BA.2, BA.4/BA.5 strains, with EC50 values of 14.25, 11.94, 53.66, 69.55, 33.70 and 20.98 ng/ml, respectively (Fig. 3C).

Figure 3.

Biological characteristics of ISH0339. (A) ELISA binding (OD450) reactivity of ISH0339-85, ISH0339-151 and ISH0339 to BA.4/BA.5 RBD. (B) Plot showing ELISA binding (OD450) reactivity of ISH0339-85, ISH0339-151 and ISH0339 to BA.4.6/BF.7 RBD. (C) ELISA binding (OD450) reactivity of ISH0339 to SARS-CoV-2 RBD of Wild Type strain, variants Delta, BA.1, BA.1.1, BA.2 and BA.4/BA.5. (D) The neutralizing activity of ISH0339 against pseudovirus for Delta, BA.1, BA.2 and BA.4/BA.5. (E) The blocking activity of ISH0339 against BA.2.75 RBD. (F) The blocking activity of ISH0339 against BQ.1.1 RBD.

Pseudovirus and authentic virus neutralization assay of ISH0339

To characterize the broad-spectrum neutralization activity of ISH0339, we performed neutralization assays with both pseudovirus and authentic virus against emerging variants. ISH0339 exhibited potent neutralization activities against Delta, BA.1, BA.2, BA.4/BA.5 in pseudovirus neutralization assay, with IC₅₀ values of 0.235, 0.028, 0.086 and 0.089 nM, respectively (Fig. 3D). We have further validated that ISH0339 showed strong blocking activity against latest Omicron BA.2.75 and BQ.1.1 subvariants, with IC₅₀ values of 1.49 and 14.18 μg/ml, respectively (Fig. 3E and F). ISH0339 displayed similar neutralization trends in the authentic neutralization assay. Specifically, ISH0339 neutralized the authentic BA.2 and BA.5 with IC₅₀ of 2.0 and 3.9 ng/ml, respectively (Table 1). Taken together, our data demonstrated that ISH0339 as bsAb format can leverage the synergistic effects of parental mAbs for broad binding and neutralization activities across all emerged variants of concern.

Table 1.

ISH0339 neutralized the authentic BA.2 and BA.5 virus

Neutralizing activity of ISH0339 against authentic BA.2 virus
sample	Concentration (ng/ml)
	500	250	125	62.5	31.3	15.6	7.8	3.9	2.0	1.0
ISH0339	3/3 (100%)	3/3 (100%)	3/3 (100%)	3/3 (100%)	3/3 (100%)	3/3 (100%)	3/3 (100%)	3/3 (100%)	3/3 (100%)	0/3 (0%)
Neutralizing activity of ISH0339 against authentic BA.5 virus
sample	Concentration (ng/ml)
	500	250	125	62.5	31.3	15.6	7.8	3.9	2.0	1.0
ISH0339	3/3 (100%)	3/3 (100%)	3/3 (100%)	3/3 (100%)	3/3 (100%)	3/3 (100%)	3/3 (100%)	2/3 (66.7%)	0/3 (0%)	0/3 (0%)
PC	CPE was observed in all the wells
NC	The cells grew well without CPE

PC: the virus suspension is incubated with the cells.

NC: the PBS is incubated with the cells.

Binding epitopes prediction and mechanism of action of ISH0339 on SARS-CoV-2

To dissect the potential mechanism responsible for such potent and neutralization activities of ISH0339, bioinformatic analysis were performed to predict the binding epitopes of ISH0339 on SARS-CoV-2. ISH0339-85 exhibited distinct binding affinity of BA.1 and BA.1.1, while having no neutralization activity against BA.4.6/BF.7. On the other hand, ISH0339-151 showed similar neutralizing activities against Delta, BA.1 and BA.2, but weaker neutralizing affinity against BA.4/BA.5. Based on bioinformatical analysis, we investigated the mutated amino acids within the RBD region of above strains (Fig. 4A). For ISH0339-85, the RBD sequences between BA.1 and BA.1.1 differed solely in the R346K. The complete loss of neutralizing activity of ISH0339-85 against BA4.6/BF.7 further validated the binding epitope, as they bear the R346T mutation. For ISH0339-151, the differences between Delta, BA.2 and BA.4/BA.5 in the RBD sequences mainly focus on the amino acids of neutralizing epitopes of Class I (S477, T478, E484, F486) and Class II (L452, N460, Q493, G496) [16, 23]. Since ISH0339-151 had lower neutralizing affinity against BA.4/BA.5 compared with Delta and BA.2, we identified the key binding sites of ISH0339-151 to be Class I epitopes, especially amino acid F486 and surrounding residues, as BA.4/BA.5 and Delta differed mainly in Class I epitopes while Delta and BA.2 shared similarities mainly in Class I epitopes (Supplementary Table 3). Therefore, the key binding site for ISH0339-85 on BA.1 RBD is predicted to be R346 of Class III epitopes, while for ISH0339-151 are Class I epitopes (N477, K478, A484, F486) on BA.1 RBD. Previous studies reported four classes of neutralizing regions on the RBD protein (Fig. 4B) [16]. After alignment we identified the binding epitopes of ISH0339-85 and ISH0339-151 belonged to neutralizing epitopes Class III and Class I, respectively. We further predicted the interaction sites between SARS-CoV-2 BA.1 RBD with ISH0339-85 or ISH0339-151 by structural docking using AlphaGo2 (DeepMind Technologies) and refined with Rosetta (Apple Inc.). In the docking process, when interconnecting with antibody ISH0339-85 or ISH0339-151, the RBD area is limited to amino acids 330–360 (Class III) or 420–490 (Class I), respectively, consistent with the binding and neutralization studies (Supplementary Fig. 3). Therefore, the main neutralization mechanism of ISH0339 was the dual-site cooperativity which completely blocks the virus binding and receptor recognition (Fig. 4C). At the same time, ISH0339 with four binding epitopes that can simultaneously neutralize multiple viruses was also an important virus clearance mechanism (Fig. 4C). Future X-ray crystallography or cryo-electron microscopy structural studies will be needed to validate the binding sites and detailed mechanism of actions. We further conclude that ISH0339 will also provide protection against the most-newly emerged BQ.1 and BQ.1.1, as the mutation sites in those subvariants were still within the targeting scope of ISH0339 (Fig. 4A).

Figure 4.

Epitope prediction to dissect the broad and potent neutralization mechanism of ISH0339. (A) Bioinformatical analysis for multiple SARS-CoV-2 strain S protein mutations, with RBD annotated by the red box. (B) Neutralization epitope classes in the SARS-CoV-2 RBD. Different classes are colored according to their architecture (red, Class I of neutralization epitope; blue, Class II of neutralization epitope; yellow, Class III of neutralization epitope; green, Class IV of neutralization epitope; gray, SARS-CoV-2 RBD). (C) Mechanism of neutralizing actions of ISH0339. Different modules are colored according to their architecture (green, Constant region of ISH0339-151; blue, Variable region of ISH0339-151; yellow, scFv of ISH0339-85; gray, SARS-CoV-2 RBD; orange, Human ACE2; light yellow, epitope 2 on RBD of ISH0339-85; red, epitope 1 on RBD of ISH0339-151).

ISH0339 protects against SARS-CoV-2 omicron BA.2 virus infection in vivo

We further assess the protection efficacy of ISH0339 for both prophylactic and protective purpose in a mesocricetus auratus challenging model (Fig. 5A). In the pre-exposure prophylaxis study, hamsters were treated with nasal spray of 50 mg/kg ISH0339, and 2 h later challenged with 1 × 10⁴ median tissue culture infectious dose (TCID50) of BA.2 virus. In the post-exposure therapeutic study group, after 2 h of BA.2 virus challenge, hamsters were treated with 25 and 50 mg/kg ISH0339 through intraperitoneal administration. Both the prophylactic group and therapeutic group displayed substantially reduced viral titers in the lung compared to the PBS control (Fig. 5B and C). In the therapeutic group, the lung viral load was significantly reduced by 2.4-log and 1.7-log in high-dose and low-dose group, respectively (Fig. 5B). In the prophylactic study, the viral load in lung tissues was decreased by 1.5-log with almost no virus detection in four hamsters (Fig. 5C). There were some discrepancies in protection efficiency in the prophylactic group, which might be due to the inhalation or exhalation condition of the animal when the nasal spray was administered. To reduce this experimental error, microsprayer aerosolizer system that can atomize the liquid sample into aerosol directly and quantitatively in the animal is recommended for future studies.

Figure 5.

Protection efficacy of ISH0339 against Omicron BA.2 in mesocricetus auratus. (A) Administration strategy of ISH0339 in the mesocricetus. Prophylaxis and therapeutic effects of ISH0339 in the hamster model were performed in study A (n = 8) and study B (n = 8). (B) The SARS-CoV-2 viral load of therapeutic group with 25 mg/kg and 50 mg/kg of ISH0339 in hamster lungs. (C) The SARS-CoV-2 viral load of prophylaxis group using 50 mg/kg ISH0339 in hamster lungs. Data are shown as means with s.d. and were analyzed by one way analysis of variance (**P < 0.01; ****P < 0.0001; n.s. not significant).

Pharmacokinetic analysis of single dose of ISH0339

Pharmacokinetic analysis of ISH0339 was undertaken in rats following single IV bolus at 25, 50, 100 and 300 mg/kg or intranasal administration at 26.5 and 53.0 mg/kg. For the IV bolus administration of ISH0339 at 25, 50, 100 and 300 mg/kg, the time to maximum serum concentration (T_max) of ISH0339 was 0.083–2.055 h, and the terminal half-lives (T_1/2) of ISH0339 were as long as 240.96–324.38 h (Fig. 6A,Supplementary Table 4). ISH0339 showed no sex differences at all dose levels when comparing the AUC0-last and Cmax in female and male rats. For the intranasal administration of ISH0339 at 26.5 and 53.0 mg/kg, respectively, the serum concentrations were below the lower limit of detection, indicating that ISH0339 from nasal spray will not enter blood circulation (Fig. 6B).

Figure 6.

Pharmacokinetic profile and biodistribution tracking of ISH0339. (A) Serum concentration of ISH0339 in Sprague–Dawley rats following single dose of 25, 50, 100, 300 mg/kg by IV bolus. (B) Serum concentration profiles of ISH0339 in Sprague–Dawley rats following single dose of 26.5 and 53.0 mg/kg by intranasal administration. (C) Quantification of the average radiant efficiency of different organs at 96 h after nasal delivery of ISH0339 (1, 3, 10 mg/kg).

Biodistribution tracking of ISH0339 in mice after nasal spray

To determine the biodistribution of ISH0339 in mice after intranasal administration, different doses of Alexa Fluor 750 labeled ISH0339 were delivered, and the levels of fluorescence-labeled antibody were determined at different time points after administration. Whole-body images showed that 2 min, 16 min and 2 h after intranasal administration, ISH0339 were mainly distributed in the nasal passages for the 1, 3, 10 mg/kg groups (Supplementary Fig. 4).

We further assessed the ex vivo distributions of ISH0339 in mice after intranasal administration. The fluorescence signals in mice after administration were imaged and quantified. We found that ISH0339 was mainly distributed in the lungs, followed by the nasal cavity at 2 h for the different dosing groups (Supplementary Fig. 5A). The antibody levels maintained in the lungs and nasal cavities at 24 h (Supplementary Fig. 5B) and decreased at 48 h post administration (Supplementary Fig. 5C). The antibodies could still be detected in the lungs at 96 h after administration (Fig. 6C), which provided evidence for long-term protection against SARS-CoV-2 by nasal spray of ISH0339.

Extended toxicity analysis of single-dose ISH0339

The rats received ISH0339 at single doses of 50, 100, 300 mg/kg/dose by IV bolus or 30 and 100 mg/kg by intranasal administration following a 2-week recovery period. All rats survived to the end of the experiment. There were no test article-related changes in body weight, food consumption, ophthalmology, body temperature, clinical pathology (hematology, serum chemistry, coagulation and urinalysis), immunophenotyping analysis, gross necropsy, organ weights and histopathology. Therefore, the no-observed-adverse-effect level for male and female rats was 300 mg/kg/dose for IV injection and 100 mg/kg for intranasal administration, respectively.

DISCUSSION

In the present study, we described the in vitro activity, in vivo efficacy, pharmacokinetics and toxicity profiles of ISH0339, a novel tetravalent broadly neutralizing bispecific antibody against SARS-CoV-2 with long-term protection efficacy. ISH0339 was composed of two non-competing parental mAbs targeting distinct epitopes on SARS-CoV-2 RBD to avoid viral escape, and with engineered Fc for prolonged half-life for long-term protection. Through biological functional characterization, ISH0339 showed an excellent broad spectrum of binding and neutralizing activities especially for the newly emerging SARS-CoV-2 variants, including Delta, BA.1, BA.2, BA.4, BA.5, BA.4.6, BF.7, BA.2.75 and BQ.1.1, which escaped immunity of most existing vaccines and therapeutics. In particular, the ability to neutralize the live virus was outstanding. ISH0339 neutralized the authentic BA.2 and BA.5 virus with effectively protecting concentration of 2.0 and 3.9 ng/ml, respectively, which was more powerful than the previously reported neutralizing antibodies [19]. Moreover, half-lives of ISH0339 were identified as 261.75–397.75 h in rats. Furthermore, following single IV or intranasal administration of ISH0339 into Sprague–Dawley rats, no obvious adverse effects were observed.

A panel of mAbs targeting the SARS-CoV-2 RBD protein have been developed and used in treatment or prophylaxis against COVID-19 [7, 20, 21]. However, most of the newly emerged SARS-CoV-2 variants have escaped the authorized monoclonal neutralizing antibodies [24, 25]. The development of new anti-SARS-CoV-2 drugs should bear the properties of broad-spectrum and long-term protection. Several studies have been proved that bsAbs exhibit enhanced breadth and potency than parental mAbs in anti-infection drug development and could be effective against viral immune escape [21, 22]. ISH0339 was designed as a tetravalent broadly neutralizing bispecific antibody and two delivery approaches were developed, IV injection for treatment and nasal spray for prevention. The rationale for broad-spectrum protection and avoiding viral immune escape of ISH0339 as a bsAb is that it is unlikely that one mutation in the RBD protein will simultaneously render ineffectiveness of both parental mAbs targeting completely different epitopes. Notably, even though the two parental mAbs of ISH0339-85 and ISH0339-151 could neutralize most variants of concern (VOCs), some subvariants still evaded the neutralization of each parental mAb, including BA.1.1 and BA.4.6/BF.7 for ISH0339-85 and BA.4/BA.5 for ISH0339-151. Instead, as presented in the epitope prediction and bioinformatical analysis, ISH0339 cooperatively targeted two distinct neutralizing epitopes of Class III and Class I and maintained efficient neutralization of all emerged VOCs. Moreover, tetravalent ISH0339 with four binding sites that can simultaneously neutralize multiple viruses was also an important virus clearance mechanism. We hypothesized that ISH0339 could also be protective against future emerging SARS-CoV-2 subvariants without simultaneous mutations in both Class I and Class III epitopes, and more studies will be needed to validate this point.

Another advantage of ISH0339 is that it could provide long-protection in both delivery strategies. For preventive purpose, the administration of ISH0339 via nasal spray could be convenient to use in daily scenario. The high expression level, excellent purification yield and good formulation stability from the commercial stable cell line and process development for ISH0339 further supports the daily use as nasal spray agents (data not shown). Meanwhile, this dosage form ensured that most of the antibodies remain in the nasopharynx without entering the bloodstream for the safety of the biological agent. A prolonged half-life mutation (YTE) in the Fc region of ISH0339 would also have an explicit advantage [26, 27]. In this study, ISH0339 with prolonged blood residency would also provide long-lasting effects in the treatment SARS-CoV-2 infection, especially for the elder people or immune compromised individuals.

In summary, we report here the development of ISH0339, a novel tetravalent broadly neutralizing bispecific antibody that binds specifically to distinct epitopes of SARS-CoV-2 with prolonged antibody half-life. Preclinical characterization including pharmacology, pharmacodynamics, pharmacokinetics and toxicology studies showed that ISH0339 has the potential to be a novel agent for the treatment and prevention of SARS-CoV-2.

FUNDING

This work was supported by SunHo (China) BioPharmaceutical Co., Ltd (ISH0339), and Training Program of the Major Research Plan of the National Natural Science Foundation of China (92269118), and Scientific Research Project of Jiangsu Health Commission (M2022013).

CONFLICT OF INTEREST STATEMENT

Huabing Yang, Dongcheng Jiang, Jihong Chen, Xiaoling Jiang, Chunling Qin, Zhenzhen Huang, Chongbing Wu, Ying Zhou, and Liusong Yin are employees of SunHo (China) BioPharmaceutical Co., Ltd.

ETHICS AND CONSENT STATEMENT

Not applicable.

DATA AVAILABILITY STATEMENT

The data underlying this article are available in the article and in its online supplementary material.

ANIMAL RESEARCH STATEMENT

All experiments were performed with the approval of the Kunming Institute of Zoology, Chinese Academy of Sciences (Reference number IACUC-RE-2022-06-001 for live SARS-CoV-2 virus challenging study in the animal biosafety level 3 facility), Nanjing Clinbridge Biotech Co., Ltd (Reference number AP-C221010 for biodistribution study) and Medicilon Preclinical Research (Shanghai) LLC (Reference numbers 19292-22007 for pharmacokinetics study, and 19292-22010 for extended toxicity study) IACUC. Care was taken throughout the study to prevent any undue animal suffering.

AUTHOR CONTRIBUTIONS

Huabing Yang (Conceptualization-Lead, Data curation-Lead, Formal analysis-Lead, Writing—original draft-Lead), Yuxin Chen (Conceptualization-Lead, Data curation-Lead, Formal analysis-Lead, Writing—original draft-Lead), Dongcheng Jiang (Conceptualization-Lead, Data curation-Lead, Formal analysis-Lead), Xiaoli Feng (Data curation-Lead, Investigation-Lead, Methodology-Lead), Ying Xu (Data curation-Supporting, Formal analysis-Supporting, Investigation-Supporting, MethodologySupporting, Software-Lead), Jiayu Wei (Data curation-Supporting, Formal analysis-Supporting, Methodology-Supporting, Software-Lead), Qingcui Zou (Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Project administration-Equal), Qiaojiang Yang (Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Methodology-Equal), Jihong Chen (Data curation-Equal, Formal analysis-Equal, Methodology-Equal, Writing—original draft-Equal), Xiaoling Jiang (Conceptualization-Equal, Funding acquisition-Equal, Methodology-Equal, Project administration-Equal), Chunling Qin (Data curation-Equal, Methodology-Equal, Validation-Equal), Zhenzhen Huang (Data curation-Equal, Formal analysis-Equal, Methodology-Equal), Chongbing Wu (Data curation-Equal, Formal analysis-Equal, Methodology-Equal, Project administration-Equal), Ying Zhou (Data curation-Equal, Formal analysis-Equal, Methodology-Equal), Minghua Li (Conceptualization-Supporting, Investigation-Supporting, Methodology-Supporting, Resources-Lead, Supervision-Supporting), Liusong Yin (Conceptualization-Lead, Data curation-Lead, Formal analysis-Lead, Funding acquisition-Lead, Investigation-Lead, Methodology-Lead, Project administration-Lead, Resources-Lead, Supervision-Lead, Validation-Lead, Writing—original draft-Lead, Writing—review & editing-Lead).