Fully human antibodies from transgenic mouse and phage display platforms
Introduction
Köhler and Milstein's invention of the hybridoma method for generating antigen specific mouse monoclonal antibodies (MAbs) opened the door for the development of antibody-based drugs. However, because rodent MAbs comprise foreign protein sequences, most of the early drugs that entered clinical development elicited immune responses from human patients [1]. Advances in molecular biology, involving the manipulation of gene sequences in vitro, and the expression of these manipulated sequences in bacterial, fungal, and mammalian cell culture systems, provided methods for re-engineering rodent antibodies to partially replace the rodent antibody sequences with functionally equivalent human amino acid sequences, thus reducing the overall immunogenicity without destroying the recognition properties of the original antibody. These methods for re-engineered rodent MAbs proved to be very successful. For example, an engineered chimeric Fab fragment comprising mouse variable and human constant region sequences was immunogenic in only 1% of human patients compared with 17% for the original mouse Fab [2]. In 1994, this Fab fragment, abciximab, became the first, of over a dozen, marketed engineered antibody products [3]. Shortly after the introduction of re-engineered MAbs, new technologies were developed that allowed for the discovery of novel MAbs derived directly from human immunoglobulin gene sequences. These new technologies can be binned into two distinct categories: in vitro assembled recombinant human antibody libraries and transgenic mice comprising human germline configuration immunoglobulin gene sequences. The first of these categories (reviewed by Hoogenboom [4]) includes libraries of recombinant antibodies cloned into bacteriophage (phage) or yeast such that the antibody variable regions are displayed on the surface, providing a ‘phenotype’ that can be used to select for, and identify, the ‘genotype’ of the recombinant antibody clone. The impact of the host is highlighted by a comparison of the selected repertoire obtained from a single library using either phage or yeast display [5•]. Antibody variable region sequences selected from the phage library represented only a subset of the clones obtained from the yeast library, and those variable region sequences not represented in the phage library were found to be poorly expressed in E. coli. Proteins such as antibodies, or antibody fragments, that are derived from eukaryotic organisms are often difficult to express within a prokaryotic cell, and this could be an important limitation for phage display. In addition to phage and yeast display, methods have been developed for directly selecting antibody sequences in vitro through the isolation of ternary complexes of mRNA, ribosome, and nascent peptide [4]. While all of these display technologies show great promise, yeast display and ribosome display are more recent, and have not had time to have an appreciable impact on existing clinical stage drug pipelines. This review will therefore focus on drugs derived from the more mature phage display methodologies. The second category of technologies for direct discovery of human sequence antibodies, transgenic mice (reviewed by Lonberg [6]), has emerged more recently than phage display, nevertheless, it has made a considerable impact on clinical stage pipelines, providing over 50 different drugs now in human clinical trials (supplemental material).
Section snippets
Phage display platforms
In 1985, Smith [7] discovered that foreign DNA sequences could be cloned into filamentous bacteriophage such that the cloned sequences are expressed on the surface of the phage particles as fusion proteins. These phage particles could then be enriched for specific sequences on the basis of the binding properties of the displayed fusion proteins. This discovery was combined with PCR amplification methods developed in the late 1980s for cloning expressed immunoglobulin variable region cDNA
Transgenic mouse platforms
In 1994, two papers reported the generation of genetically engineered mice that expressed fully human antibody repertoires that could be accessed by conventional hybridoma technology (Figure 1 [13, 14]). These engineered animals comprised targeted disruptions of the endogenous mouse heavy and κ light chain genes together with introduced transgenes comprising unrearranged human heavy chain and κ light chain gene segments. The unrearranged gene segments included in these original transgenes
Fully human MAbs in the clinic
Laboratory experience with phage display and transgenic mouse platforms, and with the antibodies derived from them, can only support a priori arguments for their usefulness as a source of well tolerated and effective human therapeutics. Now that actual clinical data on phage display and transgenic mouse derived MAbs are beginning to appear in the literature, it is possible to begin an a posteriori analysis. Some of these data are outlined below, with an emphasis on immunogenicity,
Clinical experience with fully human antibodies targeting TNFα
In 2002, adalimumab, a phage display derived antibody that neutralizes tumor necrosis factor-α (TNFα), which is associated with a variety of inflammatory diseases became the first marketed fully human MAb product [16]. It is now approved for multiple indications, including rheumatoid arthritis, Crohn's disease, and plaque psoriasis. There are two other marketed MAbs that target TNFα: infliximab, a mouse human chimeric antibody, and certolizumab pegol, a CDR grafted Fab fragment that is
Clinical experience with fully human antibodies targeting IL-12/IL-23
The cytokines IL-12 and IL-23 are heterodimeric proteins that share a common subunit, p40. IL-12 is associated with TH1 mediated immune responses, while IL-23 is associated with TH17 responses. The phage display derived antibody ABT-874 [24, 25] and the transgenic mouse derived antibody ustekinumab [26•] are each directed against p40 and neutralize both IL-12 and IL-23. In a phase 2 psoriasis trial, 237 patients received ustekinumab for up to four weekly 90 mg subcutaneous doses [26•]. During
Clinical experience with fully human antibodies targeting EGFR
The epidermal growth factor receptor (EGFR) is a tyrosine kinase signaling receptor that is upregulated on a variety of epithelial cancers. In 2006, panitumumab, which inhibits EGFR signaling [30, 31, 32, 33, 34, 35, 36], became the first marketed MAb derived from a transgenic mouse platform. There has been no direct comparison of the safety and efficacy of panitumumab and cetuximab, the already marketed chimeric anti-EGFR MAb [37]; however, the transgenic-derived MAb does appear to be less
Clinical experience with fully human antibodies targeting CD20
Expression of the CD20 antigen is restricted to B lineage lymphocytes and neoplasms derived from these cell types, making it an attractive target for both B cell lymphomas and B cell mediated autoimmune disease. The transgenic mouse derived IgG1 ofatumumab [43, 44, 45•] and the mouse–human IgG1 chimeric MAb rituximab [46] – which is currently approved for treatment of non-Hodgkin's lymphoma (NHL) and rheumatoid arthritis – both bind CD20 but recognize distinct epitopes [45•, 47]. The human
Clinical experience with fully human antibodies targeting CD4
Zanolimumab is a transgenic mouse derived human IgG1 antibody directed against the T cell antigen CD4. In an 85 patient placebo-controlled, phase 2 trial in psoriasis, no patients developed anti-drug antibodies. In phase 2 trials, in cutaneous T cell lymphoma patients (mycosis fungoides and Sezary syndrome), only 1 of 47 patients had measurable anti-drug antibodies [48]. The anti-zanolimumab titer was very low in this patient, and did not appear to be neutralizing, as demonstrated by continued
Clinical experience with fully human antibodies targeting CTLA-4
CTLA-4 (CD152) is a negative T cell signaling molecule that binds to the two ligands CD80 and CD86. This molecule is a potential target for cancer therapy based on the CTLA-4 blockade induced upregulation of immune responses to tumor cells. There are two transgenic mouse derived MAbs directed against CTLA-4, the IgG1 MAb ipilimumab [49], and the IgG2 MAb tremelimumab [50], in phase 3 clinical testing. Neither of these fully human antibodies elicits strong anti-drug antibody responses, which is
Clinical experience with fully human antibodies targeting TRAIL-R1/TRAIL-R2
Tumor necrosis factor related apoptosis inducing ligand (TRAIL) induces apoptosis by binding to the receptors TRAIL-R1 and TRAIL-R2. Two phage display derived antibodies, mapatumumab, which targets TRAIL-R1, and lexatumumab, which targets TRAIL-R2, are now in phase 2 clinical trials. Both of these drugs have agonist activity and demonstrate tumor cell killing in vitro and in preclinical animal models [57, 58]. Results from phase 1 and 2 studies have been reported for mapatumumab [59, 60]. In a
Clinical experience with fully human antibodies targeting EpCAM
EpCAM (CD136) is a widely expressed epithelial cell surface glycoprotein that is upregulated on human adenocarcinomas and squamous cell carcinoma. Several different MAbs directed against EpCAM have been tested in human patients, and a mouse MAb, edrecolomab, was previously marketed in Germany [63]. Normal epithelial cell expression of EpCAM appears to lead to dose-limiting pancreatitis for high affinity MAbs. To avoid toxicity, a low affinity (KD ∼200 nM) MAb, adecatumumab, was selected from a
Clinical experience with fully human antibodies targeting RANKL
Receptor activator of nuclear factor-κB ligand (RANKL) is a TNF family member that stimulates the maturation and activation of osteoclasts, which mediate bone resorption. The transgenic mouse derived, human IgG2 antibody, denosumab blocks RANKL activity to inhibit bone resorption. A recent review listed 21 different clinical trials for the drug, including 10 ongoing phase 3 studies for treatment of bone loss in postmenopausal women, and in cancer patients with treatment induced bone loss or
Concluding remarks
At least 66 different therapeutic drugs, derived from either phage display or transgenic mouse platforms, have entered human clinical testing. The reported binding affinities for these MAbs range over five orders of magnitude; however, most are subnanomolar, and nearly half of the MAbs from each platform have affinities in the 0.1–1 nanomolar range, with similar representation of each platform on either side of that range. Thus, the published literature does not appear to distinguish fully the
Conflict of interest statement
The author has a financial interest in Medarex, which in turn has a financial interest in many of the drugs discussed in this article.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgement
I thank Michelle Temple for assistance with the preparation of the manuscript.
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