Monoclonal Antibodies As Neurological Therapeutics Part 2

4.2. Indirect or Immune-Mediated Actions

Conserved differences in the constant regions (Fc) of IgG antibodies distinguish them into four subclasses: IgG1, IgG2, IgG3, and IgG4 [158,159]. 

Conservative differences refer to differences in individual performance in memory, thinking, emotion, etc. This difference may be related to a variety of factors such as age, gender, culture, and educational background. Among them, conservative differences are particularly significant in memory.

The relationship between conservative differences and memory is mutually influential. People with strong conservatism generally pay more attention to history, traditions, rules, etc., which can help them remember better because memory requires attention to details and context. At the same time, conservatism is also related to maintaining habits and stabilizing emotions, which also helps to improve memory.

On the other hand, conservatism does not necessarily lead to excellent memory performance. Some highly open people are quick-thinking and curious, and can understand and remember information more deeply through diversified thinking and multi-angle analysis. They are more willing to try and fail and accept challenges. This "openness" can bring more learning opportunities and memory experience, thereby enhancing memory ability.

In general, regardless of the strength of conservatism, it is important to develop one's strengths and make full use of one's advantages to improve memory. In daily life, you can stimulate the brain and enhance memory and learning ability through a variety of ways such as reading a lot, listening to music, and learning new skills. At the same time, maintaining an optimistic attitude is also a key factor in improving memory, because emotions are closely related to memory. Only by always being optimistic can we achieve better performance in all aspects. It can be seen that we need to improve memory, and Cistanche can significantly improve memory, because Cistanche has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidation and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche can also promote the growth and repair of nerve cells, thereby enhancing the connectivity and function of neural networks. These effects can help improve memory, learning ability, and thinking speed, and can also prevent the occurrence of cognitive dysfunction and neurodegenerative diseases.

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These Fc regions are involved in binding to Fe receptors (FcyR), complement factor component 1g (C1q), and the neonatal receptor (FcRn), and as a result, they determine the ability of different IgGsubclasses to mediate effector functions such as phagocytosis, antibody-dependent cell-mediated cytotoxicity, complement activation and determine their half-life and capacity for transplacental transport and transport through mucosal surfaces [159] Most unconjugated antibodies bear a human IgG1 Fc, an isotype that efficiently activates the immune system with the scope of harnessing different immune cells and molecules towards target cell killing. 

Thus, IgGl mAbs may activate natural killer (NK) cells through CD16A, induce antibody-dependent cytotoxicity (ADCC), bind to macrophage CD16A, CD32A, and CD64to promote antibody-dependent phagocytosis (ADPh), and activate the complement leading to complement-dependent cytotoxicity (CDC) [158]. More specifically, to trigger ADCCthe Fc binding domain of an antibody binds to a specific antigen expressed on the surface of aa target cell. 

The antibody is then able to recruit NK cells to lyse the target cell [150]. CDC is triggered when the C1 complement factor binds an IgG1 or IgG3 antibody-antigen complex, resulting in the activation of the complement cascade culminating in the formation of the C5b-9 membrane attack complex (MAC) forming a water pore in the target cell leading to its lysis [160]. 

Most of the marketed mAbs such as alemtuzumab and rituximab belong to the IgG1 subclass and are shown to trigger ADCC and CDC [73,161]. The immune-mediated mode of action of mAbs is schematically presented in Figure 2. 

On the other hand, IgG2 and IgG4 subclasses exhibit a lower affinity to the Fcγ receptor and are commonly preferred for blocking antigen function. More specifically, the IgG2 subclass is commonly selected to neutralize soluble antigens without inducing host effector mechanisms as in the case of erenumab and fremanezumab [154,155]. 

Similarly, IgG4 such as natalizumab and galcanezumab represent an important subclass of mAbs commonly selected when the recruitment of the host effector mechanisms is not desirable [55,155,159,162].

Figure 2. Mechanisms of action of monoclonal antibodies. MAbs may act through direct (a,b) or indirect mechanisms (c). 

The direct mechanisms include: (a) blocking ligand-receptor interactions through binding to (i) a soluble ligand or receptor or (ii) to a cell-bound ligand or receptor leading to inhibition of downstream signaling events, (b) agonism through binding to a receptor by mimicking its natural ligand leading to the activation of signaling pathways. 

Indirect mechanisms are immune-mediated as they involve the activation of certain types of immune cells and molecules to kill target cells (c). 

Most mAbs bear a human IgG1 Fc region that can activate effector cells, such as natural killer (NK) cells to induce antibody-dependent immune cell cytotoxicity (ADCC), or macrophage-inducing antibody-dependent phagocytosis (ADPH), through the interaction with their FCγ receptors. Moreover, the Fc region of mAbs can activate the complement leading to complement-dependent cytotoxicity (CDC).

4.3. Conjugated mAbs

Conjugated mAbs are combined with a drug or a radioactive substance. These mAbs are currently used in oncology to deliver these substances directly to cancer cells [163]. 

They are specifically designed to induce either a block in proliferation or direct cell death (usually apoptosis) and can deliver higher concentrations of cytotoxic agents directly to the target cells without affecting normal cells, thus reducing the potential for adverse reactions [158]. 

Ibritumomab tiuxetan is an example of a radiolabeled mAb against CD20, (a B cell surface protein), which is conjugated with radioactive Yttrium-90 and used in radioimmunotherapy and Ado-trastuzumab emtansine (also called TDM-1), is an antibody that targets the HER2 protein conjugated to a chemotherapeutic drug called DM1 [164,165].

Although conjugated mAbs have neither clinical nor experimental application in neurology, they could be used in the future to destroy targets or traffic medications to specific cell types.

4.4. Bispecific Monoclonal Antibodies

Bispecific mAbs are specially designed to recognize and bind to two epitopes simultaneously. Their unique structure confers an unlimited potential for novel functions. 

Combining the two distinct binding sites in a single molecule yields a compound function that is restricted both in space and time, which cannot be achieved by the administration of a mixture of two separate mAbs with the same specificity. 

Bispecific Abs can direct effectors cells to target cells, promote receptor internalization, deliver ligands to specific cell populations, simultaneously block two pathways, or promote shuttling across biological barriers [166]. The latter is particularly relevant to neurology where the blood-barrier barrier (BBB) is an obstacle to access of mAbs to the CNS. One specificity of a bispecific Abs can be used to shuttle it through the BBB (e.g., binding to the transferrin receptor) and the second specificity can bind to protein targets to block or promote a process or destroy brain tumor cells [167]. 

Two bispecific Abs are currently marketed and many others are in development. As an example blinatumomab, which is indicated for Philadelphia chromosome-negative relapsed or refractory acute lymphoblastic leukemia binds simultaneously to the CD3 protein of T cells and the CD19 protein of target neoplastic B cells. 

By binding to both proteins, it brings T effector cells near target neoplastic cells promoting their immune-mediated lysis [168]. Emicizumab is another bispecific Ab approved in the EU and US for Hemophilia A as it binds simultaneously with coagulation factors IXa and X [169]. 

Many other bispecific Abs are in clinical development for several uses [168]. No bispecific Abs are currently in use in neurological therapeutics. However, preclinical evidence holds promise for their use in neurology in the future. 

Delivery of the construct of a bispecific Ab with an LDLR-binding domain of apoB to facilitate its transfer across the BBB and promote alpha-secretase activity over beta-secretase activity thus favoring the neuroprotective APP cleavage by alpha-secretase using an adenoviral vector has shown beneficial effects in a mouse model of AD [170]. 

In addition, targeting simultaneously the angiogenic factor angiopoietin-2 (Ang-2) and translocator protein (TSPO), both of which are overexpressed in bevacizumab-treated glioblastomas, with a bispecific Ab in bevacizumab-treated rats resulted in prolonged survival [171]. 

Furthermore, another bispecific Ab targeting Ang-2 and vascular endothelial growth factor (VEGF) was also found to prolong survival in a mouse model with glioblastoma xenografts, suggesting that bispecific Abs targeting appropriate epitopes may be beneficial in neurooncology [172].

5. Doses, Routes of Administration and Pharmacokinetics

Regarding dosing, some mAbs are given in a fixed dose whilst others are given according to the patient's body weight. MAbs require parenteral administration for adequate bioavailability. 

In most cases, mAbs are administered either intravenously (e.g., natalizumab) or subcutaneously (e.g., eremumab). Some can be administered by either route (e.g., rituximab), whilst intramuscular administration has also been reported (e.g., palivizumab). 

Intravenous administration is chosen for greater and faster bioavailability and lower risk of immunogenicity whilst subcutaneous use is chosen to avoid intravenous access and facilitate self-administration [147,173]. 

Subcutaneously administered antibodies are taken up by lymphatics and their plasma concentration increases slowly over several days. Circulating mAbs leave the vasculature by hydrostatic and osmotic pressure gradients. Their affinity for the epitope of their specificity determines their retention in target tissues [173]. The half-lives of mAbs vary from hours to several weeks [174]. 

MAb half-life is largely determined by the binding of the constant fragment (Fc) of humanized and human Abs of immunoglobulin G (IgG) class to the neonatal receptor FcRn, expressed on many adult cell types [147]. 

More specifically, IgG antibodies are thought to be taken up by catabolic cells by fluid-phase endocytosis. Although, under neutral pH, FcRn has a low affinity for IgG, the endosome content is then acidified, thus increasing the affinity of the FcRn for IgG. The FcRn-IgG complex is then re-shuttled to the cell surface where the IgG is released under neutral pH [175]. 

Proteins and antibodies in the endosome that are not bound to the FcRn undergo proteolysis. This is a salvage pathway recycling and protecting IgGs from degradation therefore increasing their half-life without affecting their function. 

The half-life of IgG1, IgG2, and IgG4 is in the range of 18 to 21 days whereas the half-life of other proteins with comparable molecular weight is significantly shorter. The half-life of IgG3 mAbs, which have a lower affinity for the FcRn is approximately 7 days. 

Mabs that are Fc-deficient typically have an even shorter plasma half-life (e.g., 1.25 ± 0.63 h for blinatumomab in vivo), as they lack protection from degradation by the neonatal Fc receptor (FcRn) and in some cases also have a lower molecular weight than IgG, further increasing elimination through the kidneys [147,174]. 

It is conceivable that mAb internalization and FcRn-regulated release may affect the efficacy of an mAb if the dose of administration does not ensure that its free circulating fraction suffices to exert its action. Accordingly, blockade of the FcRn is therapeutically exploited to reduce the activity of pathogenic auto-antibodies (see rozanolixizumab, nipocalimab, batoclimab, and efgartigimod in Section 6.5). 

A method to increase mAb half-life is to covalently attach a polyethylene glycol (PEG) chain to the mAb molecule (pegylation) as in the case of certolizumab pegol used for rheumatoid arthritis and Crohn's disease [176]. 

The duration of biological activity may differ substantially from their half-life because the former is primarily determined by the duration of the biological effects (e.g., the time required for a depleted cell population to recover). 

Consequently, the frequency of the administration depends on the mAb, its individual properties, and the therapeutic strategy. Generally, mAbs are administered at fixed intervals, though in some cases dosing frequency may be determined by the duration of the effect as in the case of B cell depletion with rituximab treatment in multiple sclerosis (MS) and neuromyelitis optica spectrum disorders (NMOSD), where the peripheral blood CD19+ population may be used as a surrogate marker of B cells repopulation [177]. 

A notable drawback of using mAbs for neurological diseases is their low accessibility to the CNS compartment. The normal brain-to-blood IgG concentration ratio of IV-infused mAbs is approximately 0.1%. 

The passage through the BBB could be facilitated by the use of bispecific Abs where one specificity recognizes a receptor at the BBB, which promotes transcytosis, and the other specificity recognizes a potential therapeutic target such as Aβ, tau, or tumor-specific targets (Figure 3). 

The best-studied receptors for targeting brain tissue and promoting passage through the BBB are the insulin receptor (InsR), the LDL-related protein type 1 (LRP1), and the transferrin receptor (TfR) [178,179]. 

Using bispecific Abs with BBB shuttle function has been shown to increase the brain-to-blood IgG concentration ratio of IV-infused mAbs to 2–3% [180]. Other methods to improve mAb delivery to the CNS compartment are also being explored [181]. 

Interestingly, a recent double-blind trial investigated the effects of intrathecal and intravenous administration of rituximab versus placebo on several biomarkers of B cell depletion, inflammation, and neurodegeneration in progressive MS (RIVITALISE trial; NCT01212094). 

The trial was discontinued early because at interim analysis, cerebrospinal fluid (CSF) B cells were only partially and transiently depleted and neurofilament light chain levels used as a marker of axonal damage were unchanged. 

The study identified low CSF levels of lytic complement factors and paucity of cytotoxic CD56dim NK cells as key contributors to decreased efficacy of intrathecally-administered rituximab [74].

6. Indications in Neurology

6.1. Multiple Sclerosis

MAbs have revolutionized the treatment of both relapsing and progressive forms of multiple sclerosis (MS). 

Currently approved mAbs have shown their efficacy through phase all randomized controlled trials (RCTs) and are mainly used in the highly active forms of the disease, where their benefits outweigh associated risks. Infliximab, a chimeric IgG1 mAb against tumor necrosis factor-alpha (TNF-a) was tested in a phase trial but the trial had to be prematurely terminated due to increased relapse activity under infliximab treatment  182.

The first FDA approved mAb is natalizumab, a humanized antibody directed againstx4β1 integrin (CD49d), a molecule expressed on the surface of lymphocytes and monocytesand interacting with brain endothelial VLA-4 in order to mediate their entry into theCNS parenchyma. 

Natalizumab has been a great success of the translational researchas it proved to significantly reduce the relapse rate, disability progression and magneticresonance imaging evidence of disease activity [57,178]. 

Natalizumab is currently beingused as a second line agent in the treatment of highly active or rapidly evolving severerelapsing-remitting (RRlS) with excellent overall long-term risk-benefit balance [58 .

About cytokine targets, canakinumab, a human IgG1 mAb targeting Il-12 and 23 was examined in a phase II trial in RRMS. Although canakinumab significantly reduced the annualized relapse rate and a number of gadolinium-enhancing lesions on brain MRI its efficacy was not deemed satisfactory for further development, compared to other agents [183]. 

Ustekinumab is another human IgG1 mAb targeting Il-12 and 23 tested in a phase II trial in RR-MS patients. Ustekinumab subcutaneous injections showed no effect on the cumulative number of gadolinium-enhancing lesions and the trial was terminated prematurely. 

The low concentrations of ustekinimab crossing the blood-brain barrier and its administration at a stage that may be considered past the decisive step of mobilization of a Th17 autoimmune reaction were considered possible causes of its failure [184]. 

Alemtuzumab is a humanized monoclonal antibody selectively targeting CD52. Within minutes from infusion, it depletes T and B cells through antibody-dependent cell-mediated cytolysis (ADCC) and complement-dependent cytotoxicity (CDC), which is followed by slow repopulation from hematopoietic precursor cells over several months with a distinct temporal pattern [161]. 

Alemtuzumab was the first monoclonal antibody that proved its efficacy against an active comparator (interferon-β1a) in a phase II trial [10] and two phase III trials [11,12] regarding clinical and MRI outcomes. 

It is indicated for relapsing forms of MS in patients who have had an inadequate response to two or more disease-modifying treatments (DMTs) according to the FDA [13] or for highly active relapsing-remitting MS despite treatment with at least one DMT or if the disease is worsening rapidly (EMA) [185]. Rituximab is a chimeric anti-CD 20 antigen mAb initially licensed for B-cell non Hodgkin lymphomas resistant to other chemotherapy regimens [4]. 

CD20 is a 297 a.a. membrane-associated phosphoprotein present on all B cells which include pre-B cells, immature B cells, mature B cells, memory B cells, and a small fraction of T cells but not in stem cells, pro-B cells, and plasma cells [4]. 

Rituximab depletes circulating B cells but not B cells in the bone marrow or lymph nodes [4], promoting B cell lysis via antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDD), and phagocytosis by macrophages and neutrophils [73]. 

A phase II, double-blind, a trial involving 104 patients with RR-MS assigned to either rituximab or placebo showed that patients receiving rituximab had significantly fewer total and new gadolinium-enhancing lesions on MRI, and the proportion of patients in the rituximab group which exhibited at least one relapse was significantly reduced at week 24 (14.5% vs. 34.3% in the placebo group, p = 0.02) and week 48 (20.3% vs. 40.0%, p = 0.04) [75]. 

However, a randomized controlled phase III study has not been conducted with rituximab in MS patients to date. Furthermore, rituximab was the first CD20-depleting therapy to also be examined in phase II/III trial in primary progressive MS (PPMS) patients [76]. 

Rituximab did not meet the defined primary endpoints, but this trial cleared the way for the exploration of ocrelizumab in this disease stage as it gave valuable clues regarding its efficacy in progressive disease [186]. 

Nevertheless, rituximab is extensively prescribed off-label, notably in Sweden where up to 53% of MS patients may be under rituximab [77]. Ocrelizumab is a humanized mAb approved by the FDA in 2017 for the treatment of patients with relapsing or primary progressive forms of multiple sclerosis. 

It targets the CD20 antigen on B-cells and is the only intravenous anti-CD20 antibody that has been proven safe and efficacious in two randomized controlled phase III twin trials in which it was compared to subcutaneous interferon beta-1a at a dose of 44 µg three times weekly for 96 weeks. 

A statistically significant decrease in the annualized relapse rate by 46% in trial 1 and 47% in trial 2 was observed in the ocrelizumab-treated group compared to the interferon beta-1a group. 

The percentage of patients with confirmed disability progression at 12 and 24 weeks was significantly lower with ocrelizumab and the mean number of gadolinium-enhancing lesions in T1-weighted magnetic resonance scans was 94% lower with ocrelizumab in trial 1 and 95% lower in trial 2, compared to treatment with interferon beta-1a [68]. 

Ocrelizumab is the first approved treatment for primary progressive MS as it has shown benefit in several efficacy measures including a significantly lower percentage of patients with confirmed disability progression at 12 and 24 weeks and a significantly lower percentage of brain volume loss in a phase III double-blind, placebo-controlled trial [69]. Ofatumumab was approved by the FDA for MS in 2020. 

It is another anti-CD20 mAb, B-cell depleting DMT for MS. It has proven its efficacy and safety through two phase 3 double-blind studies (ASCLEPIOS I and II) in which it was compared to teriflunomide [72]. 

Patients on ofatumumab exhibited a significantly lower annualized relapse rate in both trials and the percentage of patients with disability worsening confirmed at 3 and 6 months was also significantly lower with ofatumumab compared to teriflunomide [72]. 

Ofatumumab bears the important advantage of being the first self-administered, B cell targeting DMT in MS, delivered via an autoinjector pen, enabling patients to self-administer the treatment at home, avoiding visits to the infusion center, a particularly relevant advantage during the current COVID-19 pandemic [187].

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