Recombinant Protein spdA (spdA)

What is SP-D and what is its molecular structure?

SP-D (surfactant protein-D; also PSP-D) is a 43 kDa member of the collectin family of innate immune modulators. It is constitutively secreted by alveolar lining cells and epithelium associated with tubular structures. Human SP-D is synthesized as a 375 amino acid precursor containing a 20 amino acid signal sequence and a 355 amino acid mature region. The mature molecule is characterized by:

• A 25 amino acid N-terminal linking-region

• A 177 amino acid hydroxyproline and hydroxylysine collagen-like domain

• A 46 amino acid coiled-coil segment

• A 106 amino acid C-terminal collectin-like C-type lectin domain (CRD)

The basic functional form of SP-D is a glycosylated, disulfide-linked 150 kDa trimer with an alpha-helical coiled-coil structure and three symmetrical CRDs. Typically, SP-D forms a higher-order 620 kDa, X-shaped dodecamer through disulfide bonds at the N-terminus, allowing for sophisticated discrimination of carbohydrate patterns .

What distinguishes recombinant fragments of human SP-D (rfhSP-D) from the native protein?

The full-length native SP-D has properties that make it challenging to develop as a therapeutic agent, including varying degrees of oligomerization, limited solubilization, and potential aggregation at higher concentrations . Therefore, recombinant fragments of human SP-D have been developed as alternatives.

RfhSP-D typically contains functional domains of the SP-D molecule that retain its pathogen recognition capabilities while offering improved stability and production efficiency. A stable form of rfhSP-D can be produced using mammalian cell lines and purified using affinity chromatography with N-Acetylmannosamine (ManNAc)-coupled matrix . These fragments maintain the critical carbohydrate recognition capabilities while being more amenable to pharmaceutical development.

How does SP-D recognize and interact with pathogens?

SP-D functions through its CRD domains, which recognize specific carbohydrate patterns on microbial surfaces:

• Each CRD recognizes the hydroxides of one monosaccharide

• The trimeric structure allows for discrimination of monosaccharide patterns specific to microbial pathogens

• The dodecameric structure enables even finer discrimination between self and non-self carbohydrate patterns and facilitates binding to complex antigens

This recognition system allows SP-D to identify various pathogens, including viruses. For instance, rfhSP-D can interact with the spike protein of SARS-CoV-2, as demonstrated by docking analysis showing that three amino acid residues in the receptor-binding domain of SARS-CoV-2 spike protein interact with both rfhSP-D and the ACE-2 receptor .

What mechanisms govern SP-D's dual role in inflammatory modulation?

SP-D employs a sophisticated mechanism for regulating inflammatory responses based on the occupancy of its CRDs:

This dual mechanism allows SP-D to provide a graded response to environmental challenges, enabling the immune system to appropriately respond to different levels of pathogen threat. This explains why SP-D can both suppress inflammation under certain conditions and promote it under others.

How do genetic polymorphisms affect SP-D function?

Genetic variations in SP-D can significantly impact its function and potentially disease susceptibility. One notable polymorphism is the Met11-Thr11 transition in humans, which appears to prevent the formation of higher-order oligomers . This structural change potentially affects the ability of individuals carrying this polymorphism to interact effectively with microorganisms .

The inability to form dodecamers would impair the protein's capacity for fine discrimination of complex carbohydrate patterns and potentially alter its inflammatory modulation function through the SIRP alpha and calreticulin/CD91 pathways. These functional changes could influence susceptibility to respiratory infections and inflammatory conditions.

What is the experimental evidence for rfhSP-D's effectiveness against SARS-CoV-2?

In vitro studies have demonstrated promising antiviral activity of rfhSP-D against SARS-CoV-2:

• Molecular docking analyses predicted interaction between rfhSP-D and the receptor-binding domain of SARS-CoV-2 spike protein

• Direct and indirect ELISA confirmed inhibition of interaction between the spike protein and ACE-2 by rfhSP-D

• Treatment with 1.67 μM rfhSP-D inhibited viral replication by approximately 5.5-fold in experiments using clinical samples from SARS-CoV-2-positive cases

• This inhibition was more efficient than remdesivir (100 μM) in Vero cells

• An approximately two-fold reduction in viral infectivity was observed after treatment with 1.67 μM rfhSP-D

These findings suggest that rfhSP-D mediates a calcium-independent interaction with the receptor-binding domain of the spike protein, effectively competing with ACE-2 for binding and thereby reducing viral entry and replication.

What are the optimal expression systems and purification protocols for producing research-grade rfhSP-D?

Production of functional rfhSP-D requires careful consideration of expression systems and purification strategies:

Expression Systems:

• Mammalian cell lines are preferred for producing properly folded and functional rfhSP-D with appropriate post-translational modifications

• These systems overcome the limitations associated with producing full-length SP-D, which is prone to aggregation and instability

Purification Protocol:

• Affinity chromatography using N-Acetylmannosamine (ManNAc)-coupled matrix has proven effective for purifying rfhSP-D

• This approach leverages the carbohydrate-binding properties of the CRD domain

• The purification process must maintain protein stability and functionality while removing contaminants

Quality Control:

• Functional assays to verify carbohydrate recognition capabilities

• Endotoxin testing to ensure preparation purity

• Structural verification through techniques such as circular dichroism or dynamic light scattering

These methods have successfully produced stable rfhSP-D preparations suitable for both research applications and clinical trials.

What experimental design considerations are important when evaluating rfhSP-D in different disease models?

When designing experiments to evaluate rfhSP-D efficacy, researchers should consider:

Dose Determination:
The RESPONSE clinical trial employs a Bayesian continual reassessment method for dose escalation, testing three dose levels: 1 mg/kg/dose, 2 mg/kg/dose, and 4 mg/kg/dose, with a minimum of three participants per dose level . This methodology allows for optimal dose finding while minimizing subject exposure to potentially ineffective or unsafe doses.

Administration Timing and Frequency:
In the RESPONSE trial, participants receive three doses of rfhSP-D at 0 hours, 12 hours, and 24 hours, with the first dose administered after standard surfactant therapy . This regimen was developed based on preclinical pharmacokinetic data.

Safety Monitoring:
The trial uses a target level of dose-limiting events (DLEs) set at no greater than 20%, with events graded according to the published Neonatal Adverse Event Severity Score . Continuous recruitment and monitoring strategies allow for better characterization of the dose-response curve and safety profile.

Control Group Selection:
For viral inhibition studies, appropriate controls include both negative control samples and comparison with established antivirals like remdesivir .

What assays are most informative for characterizing rfhSP-D interactions with pathogens?

Several complementary approaches provide insights into rfhSP-D-pathogen interactions:

In Silico Analysis:

• Molecular docking studies can predict binding interfaces between rfhSP-D and pathogen proteins

• These computational approaches identify potential interaction residues for further verification

Binding Assays:

• Direct and indirect ELISA to confirm protein-protein interactions

• Surface plasmon resonance for real-time binding kinetics

• Pull-down assays to isolate protein complexes

Functional Inhibition Assays:

• Viral replication assays measuring expression of viral genes (e.g., RdRp gene of SARS-CoV-2) via quantitative PCR

• Viral infectivity assays in appropriate cell lines (e.g., Vero cells for SARS-CoV-2)

• Competitive binding assays to assess inhibition of pathogen-receptor interactions

A comprehensive characterization typically employs multiple assay types to confirm both binding and functional inhibition.

What is the current state of clinical testing for rfhSP-D in neonatal respiratory conditions?

The RESPONSE trial is currently evaluating rfhSP-D for prevention of bronchopulmonary dysplasia (BPD) in premature infants:

Trial Design:

• Single-center, dose-escalation, phase I safety study

• Target population: 24 infants born between 23+0 and 29+6 weeks gestation with respiratory distress syndrome

• Opened on February 6, 2024, with a projected 12-month recruitment period

Intervention:

• Three doses of rfhSP-D via endotracheal route at either 1 mg/kg, 2 mg/kg, or 4 mg/kg

• Administration in addition to routine surfactant replacement therapy

Primary Outcome:

• Evaluation of safety profile across dose levels

• Establishment of recommended phase 2 dose (RP2D)

Scientific Rationale:
Preclinical data demonstrated efficacy of rfhSP-D in reducing inflammation in chronic inflammatory lung disease caused by SP-D deficiency. SP-D knockout mice develop symptoms of chronic obstructive pulmonary disease and emphysema relevant to BPD, which are correctable following treatment with recombinant SP-D .

What is the potential for rfhSP-D as an antiviral therapy against respiratory viruses?

Research indicates promising antiviral activity of rfhSP-D:

Against SARS-CoV-2:

• RfhSP-D (1.67 μM) inhibited viral replication by ~5.5-fold in vitro, outperforming remdesivir (100 μM)

• Achieved approximately two-fold reduction in viral infectivity

• Functions by interfering with spike protein binding to ACE-2 receptor

Mechanism of Action:
The antiviral activity seems to work through:

• Direct binding to viral proteins (demonstrated with SARS-CoV-2 spike protein)

• Competition with cellular receptors (blocks spike protein-ACE-2 interaction)

• Potential additional immunomodulatory effects

Comparative Advantages:

• Natural immune protein with potentially favorable safety profile

• Functions at significantly lower concentrations than some existing antivirals

• Could potentially be effective against multiple respiratory viruses due to its pattern recognition capabilities

This research supports further investigation of rfhSP-D as a broad-spectrum antiviral agent, particularly for respiratory infections.

What are promising areas for expanding rfhSP-D therapeutic applications?

Beyond the current applications in neonatal respiratory conditions and SARS-CoV-2, several research directions appear promising:

• Exploring efficacy against a broader range of respiratory pathogens

• Investigating alternative delivery methods for pulmonary and systemic administration

• Developing modified variants with enhanced stability or targeting capabilities

• Examining potential benefits in chronic inflammatory lung conditions

• Combining rfhSP-D with other therapeutic agents for synergistic effects

The dual antimicrobial and immunomodulatory properties of rfhSP-D make it particularly interesting for conditions involving both infection and dysregulated inflammation.

What methodological advances would enhance rfhSP-D research?

Several technological advances could accelerate rfhSP-D research:

• Development of standardized functional assays to better compare results across studies

• Creation of improved animal models that better recapitulate human SP-D biology

• Advanced imaging techniques to visualize rfhSP-D interactions with pathogens in situ

• High-throughput screening methods to identify optimal rfhSP-D variants

• Long-term stability studies to support translation into clinical applications