Hyaluronidase, an enzyme that degrades hyaluronic acid (HA) in the extracellular matrix, has emerged as a key tool for improving the efficacy of monoclonal antibody therapies. Its ability to degrade HA, a major component of the extracellular matrix that impedes the distribution of large molecules in tissues, has opened new avenues for improving the pharmacokinetics and therapeutic outcomes of antibody-based treatments. When used in combination with monoclonal antibodies such as daratumumab (an anti-CD38 monoclonal antibody) and rituximab (an anti-CD20 monoclonal antibody), hyaluronidase can facilitate better tissue penetration, improve bioavailability and enhance overall clinical efficacy.

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Hyaluronidase: Mechanism of Action

Hyaluronidase enzymes catalyze the hydrolysis of hyaluronic acid (HA), a glycosaminoglycan found in the extracellular matrix, connective tissues and synovial fluid. By breaking down HA, hyaluronidase reduces the viscosity and density of the extracellular matrix, which increases tissue permeability and improves the diffusion and distribution of therapeutic agents, such as monoclonal antibodies, within the tissue.

The ability to modify the extracellular matrix is particularly valuable in cancer therapy because solid tumors often have a high density of HA that can act as a barrier, limiting the effective delivery of therapeutic antibodies and other large molecules. Hyaluronidase reduces this barrier, allowing monoclonal antibodies to better penetrate tumor tissue and improve drug efficacy.

Figure 1. Hyaluronidase-powered microneedles for significantly enhanced transdermal delivery efficiency. (Hu et al., 2022)

Monoclonal Antibodies in Cancer Therapy

Monoclonal antibodies (mAbs) are a cornerstone of modern cancer therapy. They are designed to target specific antigens on cancer cells, resulting in immune-mediated cell death, inhibition of tumor growth, or modulation of immune responses. Daratumumab and rituximab are two examples of monoclonal antibodies widely used in oncology, particularly in the treatment of hematologic malignancies.

• Daratumumab: Daratumumab is an anti-CD38 monoclonal antibody primarily used in the treatment of multiple myeloma. CD38 is a cell surface glycoprotein that is highly expressed on malignant plasma cells in multiple myeloma, making it an ideal target for daratumumab. Daratumumab works through multiple mechanisms, including direct cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and immune modulation.

Figure 2. Mechanisms of action of daratumumab. (Lamb, 2020)

• Rituximab: Rituximab is an anti-CD20 monoclonal antibody used to treat several B-cell malignancies, including non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL) and autoimmune diseases such as rheumatoid arthritis. CD20 is a surface protein found on B cells, and rituximab induces B cell depletion through mechanisms such as ADCC, CDC and apoptosis.

Figure 3. Rituximab-opsonized B cells are subject to attack and killing by at least three pathways. (A) Binding of rituximab causes activation of the complement cascade, which generates the membrane attack complex that can directly lyse B cells by complement-mediated cytotoxicity. (B) Complement activation also deposits C3b/iC3b fragments on the B cell. The Fc portion of rituximab and the deposited C3b/iC3b fragments allow for recognition by both Fcγ receptors and complement receptors 1 and 3 on macrophages, which leads to phagocytosis and antibody-dependent cell- mediated cytotoxicity. (C) Binding of rituximab allows interaction with natural killer cells via FcγRIII and complement receptor 3, which leads to antibody-dependent cell-mediated cytotoxicity. Abbreviations: ADCC, antibody-dependent cell-mediated cytotoxicity; FcγR, Fcγ receptor; MAC, membrane attack complex; NK, natural killer. (Taylor and Lindorfer, 2007)

Both daratumumab and rituximab are highly effective but can be hindered by challenges such as poor tissue penetration, limited bioavailability, and slow infusion times, especially in solid tumor settings or in patients with high tumor burden.

Mechanisms by Which Hyaluronidase Enhances Therapeutic Efficacy

The benefits of hyaluronidase in combination therapies with daratumumab and rituximab can be attributed to several mechanisms:

• Increased Interstitial Fluid Pressure: Hyaluronidase reduces the viscosity of the extracellular matrix by breaking down HA, lowering the interstitial fluid pressure. This makes it easier for therapeutic agents to diffuse into tissues, including those with high cellularity, such as tumors.
• Enhanced Diffusion Coefficients: By cleaving HA and altering the architecture of the extracellular matrix, hyaluronidase increases the diffusion coefficients of monoclonal antibodies. As a result, drugs such as daratumumab and rituximab can more easily reach their targets, enhancing their therapeutic effect.
• Reduced Immunogenicity and Infusion Reactions: Subcutaneous formulations containing hyaluronidase may reduce the incidence of infusion-related reactions compared with intravenous administration. This is particularly important with monoclonal antibodies such as rituximab, which are known to cause reactions such as fever, chills, and rash.

Hyaluronidase in Combination Therapies with Daratumumab

The infusion process of daratumumab can sometimes be challenging due to its relatively high viscosity, which makes it difficult to deliver by standard intravenous (IV) infusion. By combining daratumumab with hyaluronidase, the infusion process is greatly improved. Hyaluronidase breaks down the extracellular matrix, allowing daratumumab to be better distributed in the tissues. This combination has several important advantages:

• Enhanced Bioavailability and Tissue Penetration: Hyaluronidase helps break down HA in the extracellular matrix, which reduces tissue viscosity and increases tissue permeability. This results in improved tissue penetration of daratumumab, particularly in areas with high HA content such as tumors and inflamed tissues.
• Subcutaneous Administration: Traditionally, daratumumab is administered intravenously, which can be time-consuming and uncomfortable for patients. By combining daratumumab with hyaluronidase, the formulation allows for subcutaneous administration of the antibody. This method is more convenient for patients, as it is less invasive, reduces infusion times, and is associated with fewer infusion-related reactions. Moreover, the subcutaneous route allows for more consistent dosing and potentially improved patient compliance.
• Reduced Tumor Microenvironment Barrier: The tumor microenvironment often contains a dense extracellular matrix with a high concentration of HA, which acts as a physical barrier to the delivery of therapeutic agents. Hyaluronidase disrupts this matrix, allowing daratumumab to penetrate deeper into tumor tissue. This may enhance daratumumab's ability to target and eliminate myeloma cells that might otherwise evade treatment.

Hyaluronidase in Combination Therapies with Rituximab

Like daratumumab, rituximab also benefits from the addition of hyaluronidase in combination regimens. The use of rituximab is not limited to hematologic cancers; it has also been studied in solid tumors. However, the ability of rituximab to penetrate dense tumor tissue may be limited due to the high HA content in the tumor microenvironment.

• Enhanced Distribution and Efficacy: Hyaluronidase helps break down HA in the tumor matrix, allowing rituximab to reach its target cells more effectively. This is particularly important for solid tumors or tumors with dense stromal components, where rituximab's delivery is hindered by physical barriers.
• Subcutaneous Administration: Similar to its role with daratumumab, hyaluronidase may facilitate subcutaneous administration of rituximab. This route of administration is more convenient and less cumbersome for patients than the traditional intravenous route. Subcutaneous administration of rituximab has been shown to improve patient convenience, reduce infusion-related side effects and improve overall treatment compliance.
• Improved Pharmacokinetics: Combining rituximab with hyaluronidase may improve its pharmacokinetics by facilitating its distribution throughout the tissue, allowing for more effective engagement with CD20-positive B cells. By reducing the local viscosity of the extracellular matrix, hyaluronidase allows for faster and more widespread distribution of rituximab, which is critical for targeting large or otherwise inaccessible tumors.

Case Studies

Case 1: Subcutaneous rituximab with recombinant human hyaluronidase in the treatment of non-Hodgkin lymphoma and chronic lymphocytic leukemia; Hill and Davies, 2018

Rituximab, an anti-CD20 monoclonal antibody, has demonstrated significant clinical benefit in several B-cell malignancies. Originally formulated for intravenous infusion, its administration is often associated with prolonged infusion times and the risk of infusion-related reactions. To address these challenges, a clinical development program investigated subcutaneous administration of rituximab in combination with recombinant human hyaluronidase. This study summarizes the evidence showing that subcutaneous fixed-dose rituximab provides noninferior pharmacokinetics and comparable clinical efficacy in the treatment of follicular lymphoma, chronic lymphocytic leukemia, and diffuse large B-cell lymphoma. This alternative dosing regimen is preferred by both patients and healthcare providers and offers time and cost efficiencies.

Table 1. Response rates observed during the SABRINA study at the end of induction treatment showing comparable outcomes between intravenous and subcutaneous administrations across all body surface areas. (Hill and Davies, 2018)

Case 2: Subcutaneous daratumumab and hyaluronidase-fihj in newly diagnosed or relapsed/refractory multiple myeloma; Sanchez et al., 2021

Daratumumab, a CD38-targeting monoclonal antibody approved for multiple myeloma, has been reformulated from an intravenous (IV) to a subcutaneous (SC) formulation co-formulated with hyaluronidase (Dara-SC). Clinical trials, including PAVO and COLUMBA, have shown that Dara-SC has comparable efficacy, pharmacokinetics and safety to IV daratumumab, with the added benefits of significantly shorter administration time, fewer infusion-related reactions (IRRs) and a smaller injection volume. The Phase III COLUMBA study confirmed non-inferiority in relapsed/refractory multiple myeloma (RRMM), with fewer IRRs in the SC arm. The ongoing PLEIADES trial continues to evaluate Dara-SC in combination with standard of care in newly diagnosed and relapsed/refractory patients, showing promising response rates and a favorable safety profile.

Figure 4. Response and safety data in clinical trials of subcutaneous and intravenous daratumumab. d, dexamethasone; Dara-IV, intravenous daratumumab; Dara-SC, subcutaneous daratumumab; IRR, infusion-related reaction; M, melphalan; n, sample size; N/A, not applicable; ORR, overall response rate; P, prednisone; R, lenalidomide; V, bortezomib.

In summary, the use of hyaluronidase in combination with monoclonal antibodies such as daratumumab and rituximab represents a significant advance in therapeutic strategies for cancer and other diseases. By enhancing tissue penetration, improving pharmacokinetics and enabling more convenient subcutaneous administration, hyaluronidase helps overcome some of the major limitations of monoclonal antibody therapies. This combination approach promises not only to improve clinical outcomes, but also to enhance patients' quality of life, marking a step forward in the evolution of cancer treatment and biologic therapies.

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