Encyclopedia
Tumor-targeting monoclonal antibodies (mAbs) are the most widely used and characterized immunotherapy for hematologic and solid tumors.
- NK cell
- monoclonal antibodies
- glycoengineering
1. Introduction
There are over 100 monoclonal antibodies (mAbs) on the market used as cancer immunotherapeutics that have various mechanisms of action, including checkpoint inhibitors, targeted radiation or toxins, blocking cell growth, and the induction of leukocyte effector functions, among others, that have been described in recent reviews [1,2]. Over 30 Food and Drug Administration-approved mAbs target various tumor antigens. The first mAb therapy utilized for treating cancer, and that continues to be broadly used, is rituximab. This therapeutic mAb recognizes CD20 and has greatly improved the survival rate of patients with B cell malignancies, including non-Hodgkin lymphoma (NHL) and follicular lymphoma [3]. The 4-year overall survival of patients with follicular lymphoma that received the standard chemotherapy of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) was 69%, which increased to 91% for patients that received CHOP with the addition of rituximab (R-CHOP) [3,4]. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune process that contributes to the effector mechanisms of rituximab [5]. This cytolytic event is primarily mediated by natural killer (NK) cells, which are lymphocytes of the innate immune system that survey the body for transformed and virally infected cells [6]. ADCC by NK cells is mediated by CD16A, a fragment crystallizable (Fc) receptor (FcR) encoded by FCRG3A that binds to IgG [7]. CD16A-expressing NK cells represent the major subset in the peripheral blood (≈90%) and are the focus of efforts to enhance endogenous NK cell-mediated ADCC for clinical applications [8,9]. Multiple studies have been conducted to elucidate the molecular regulation and function of this receptor in NK cells and have led to the initiation of clinical trials centered around exploiting CD16A as a critical NK cell effector molecule [10]. Mechanisms to improve NK cell-mediated ADCC have been shown to enhance the efficacy of cancer therapies that rely on this process for tumor clearance. There have been several approaches aimed at increasing the binding affinity between CD16A and mAbs, including the direct modification of mAbs through amino acid substitutions or glycoengineering [11,12], as well as modifications to the Fc receptor [13,14].
4. Therapeutic mAb Engineering
Given that increased IgG binding affinity due to CD16A polymorphisms can enhance ADCC and improve clinical outcomes, a strategy has emerged to improve therapeutic mAb function by modifying the Fc portion of tumor-targeting antibodies. This has been achieved through different approaches, such as glycosylation and amino acid substitutions. Shields et al. performed alanine screening on the Fc region of IgG1 and found that the combined substitution of Ser298, Glu333, and Lys334 to alanine increased binding affinity to CD16A and resulted in augmented ADCC [11]. Other point mutations have also been shown to be effective at increasing IgG binding affinity to CD16A, including Ser239Asp and Ile332Glu, as well as Gly236Ala, Ser239Asp, Ala330Leu, and Ile332Glu (referred to as GASDALIE) [76,77]. The GASDALIE mutations increased the affinity of IgG binding to CD16A by 20-fold, while only slightly increasing binding affinity to the inhibitory FcγR CD32B [76]. Increasing affinity for CD16A while not increasing the affinity for CD32B, which could dampen activation signaling, is expected to increase leukocyte effector functions. The increase in CD16A affinity was also observed with a similar series of mutations: Ser239Asp, Ala330Leu, and Ile332Glu (SDALIE) [76,78,79]. An anti-FMS-related tyrosine kinase 3 (FLT-3) mAb with Ser239Asp and Ile332Glu mutations is part of a clinical phase I/II trial to treat patients with acute myeloid leukemia (AML) (ClinicalTrials.gov identifier: NCT02789254). Recent in vitro studies using this FLT-3 mAb suggested that it could also be suitable for the treatment of B cell acute lymphoblastic leukemia (B-ALL) [80]. A screen that aimed to elucidate increased CD16A binding/decreased off rate and also prevent increased affinity for inhibitory CD32B found that the combination of Phe243Leu, Arg292Pro, Tyr300Leu, Val305Ile, and Pro396Leu (referred to as Variant 18) achieved this [81]. The anti-HER2 mAb margetuximab contains the Variant 18 mutations and has shown promise in clinical trials, with 78% of patients evaluated showing reduced tumor size [82]. Margetuximab showed greater progression-free survival (PFS) than the non-modified therapeutic mAb trastuzumab in a phase III clinical trial for treating HER2-positive breast cancer patients (NCT02492711) [83]. These clinical outcomes demonstrate a functional benefit from increasing the affinity between mAbs and CD16A.
Glycosylation is a post-translational protein modification occurring in the endoplasmic reticulum and Golgi apparatus that regulates the structure and function of many proteins [84]. There are two primary types of linkages for oligosaccharides, N-linked and O-linked. N-linked glycans tend to be large and branched and are initiated by an N-acetylglucosamine (GlcNAc) that is attached to the nitrogen of an asparagine. N-linked glycans are added to a conserved Asn297 in the Fc region of human IgG. Structural NMR studies have shown that glycosylation alters the C’E loop which is located in the Cγ2 domain of the Fc region of IgG and this affects its binding affinity to CD16A [12]. Different types of N-glycan modifications occur, though all share a common backbone with two N-acetylglucosamine units followed by three mannose moieties [85]. An important N-glycan decoration is a core fucosylation (α1,6) at the first GlcNAc [86]. Removal of this fucose increases IgG binding affinity to FcγRs and ADCC by NK cells [87,88,89]. These studies reveal that the glycosylation of IgG1 has an allosteric impact on CD16A ligation but is not directly involved in CD16A binding [12].
Glycoengineered Anti-CD20 mAb Enhances NK Cell-Mediated ADCC
Anti-CD20 mAbs are classified as type I or type II based on glycosylation, function, and their direct biological effects. Type I mAbs, such as rituximab, induce lipid raft formation upon binding to CD20, which triggers calcium flux and caspase-mediated apoptotic signaling [90,91]. While type II mAbs also induce apoptosis, they do so in a lysosome-mediated, caspase-independent manner [92]. Obinutuzumab is a type II glycoengineered mAb that lacks the core fucose moiety on the first N-acetylglucosamine and was developed specifically to address issues of rituximab resistance in cancer patients [93,94,95]. Interestingly, a non-glycoengineered version of obinutuzumab and F(ab’)2 fragments of this mAb induced the same level of apoptosis when added to Raji cells (a B cell lymphoma cell line) as the glycoengineered antibody, suggesting that this specific function is Fc independent [92]. Type I mAbs demonstrate higher binding levels at saturating conditions compared to type II antibodies. Stoichiometry studies have shown that type I antibodies bind CD20 in a 1:1 or 2:1 (IgG:CD20) ratio, forming “seeding complexes” that enable further binding of IgG to CD20 molecules, whereas type II mAbs bind to CD20 in a 1:2 ratio, forming “terminal complexes” that do not allow the binding of additional IgG molecules [96]. This difference in numerical relationship between antibody and antigen appears to account for increased ADCC induction by type II anti-CD20 mAbs, especially in patients expressing the lower affinity CD16A (158F) variant [95]. Indeed, while obinutuzumab and rituximab both bind the same epitope on CD20, obinutuzumab demonstrated increased NK cell-dependent ADCC and IFNγ generation [97,98]. Moreover, obinutuzumab showed prolonged PFS and higher minimum residual disease (MRD) negative outcomes compared to rituximab in chronic lymphocytic leukemia (CLL) patients [99].
This entry is adapted from the peer-reviewed paper 10.3390/cancers13020312
