TRANSLATIONAL SCIENCE
TRANSLATIONAL SCIENCE From biology to therapeutics Uwe Fraass, München Since the 1980ies, when biotechnology entered medicine and the first monoclonal antibody (mab) gained regulatory approval, many advancements have been made. According to the German Association of Research Based Pharmaceutical Companies (VfA) 377 compounds were in phase II/III development in 2014. That year 59 % of all pipeline products were mabs. On average, 22% of late-stage pipeline compounds across all indications in Germany were biopharmaceuticals [1]. Amgen is engaged in the research of diverse molecular entities: mabs, therapeutic proteins, silencing messenger RNA, fusion proteins, peptides, peptidbodies, bi-specific antibodies, BITE antibody constructs (Bispecific T cell engager), antibody drug conjugates, CAR T cells (Chimeric antigen enhanced T cells), bioengineered Listeria immunotherapies and oncolytic immunotherapy viruses [2]. Biological therapy is governed by a number of key principles, some of which are illustrated below. Site-directed mutagenesis Since the mid 1990s, treatment of renal anemia with erythropoietin has become routine in dialysis patients. To take advantage of less frequent administration, less hemoglobin variability and ultimately, the option to reduce the injected protein amount, Amgen scientists tried to modify the epoetin-alfa molecule. The challenge was tri-fold: 1. To alter the amino acid sequence such that more binding sites for sialinic acid residues could be created to prolong the peptid half life; 2. To keep the unique protein folding structure to maintain Epo-receptor binding potency; 3. To mitigate antigenicity. This was achieved via semi-automated site-directed mutagenesis to alter the molecular structure in a manner to find adequate amino acid exchanges with parallel structural monitoring of the molecule (Figure 1). It yielded the invention of Darbepoetin alfa (Aranesp®) which has been used in CKD patients since 2001 [3]. r-HuEPO The principle of ‚site directed mutagenesis‘ Aranesp EDUCATION • 3 N-bound carbohydrate chains • Up to14 sialinic acid residues • 30,400 dalton • Up to 40 % carbohydrate content • 5 N-bound carbohydrate chains • Up to 22 sialinic acid residues • 38,500 dalton • Up to 52 % carbohydrate content Figure 1: The structure of r-HuEPO and Darbepoetin alfa (Aranesp®) (modified according to [3]). 42
TRANSLATIONAL SCIENCE XenoMouse® is a registered trademark of Amgen. Figure 2: Making Human Monoclonal Antibodies: Amgen’s XenoMouse® Technology XenoMouse® technology Humoral and cellular antigenicity are inherent to any introduction of a foreign protein into the human circulation. Since the invention of mabs, it has emerged that the degree of murine protein left in the administered moiety correlates with the frequency and severity of immune reactions which may give rise to mitigation or loss of its therapeutic effect as well as causing cross-reaction triggering immune reactions to human proteins. Recently, a large-scope development program of a humanized PCSK9 antibody which had remaining murine components was terminated due to intolerabilities in phase III. Accordingly, eliminating murine protein is a desirable production feature [4]. Amgen’s patented XenoMouse® hybridoma technology provides an effective system by which fully human mabs can be readily produced (Figure 2). The Xeno Mouse® strains are generated by introducing human immunoglobulin genes into mice engineered to lack functional mouse immunoglobulin genes. The result is the generation of mabs that have no mouse protein sequences and are fully human, produced via a multistep process: Step 1: Endogenous mouse immunoglobulin (heavy and light chain) gene loci are functionally inactivated in embryonic stem cells by gene-targeted deletion. These stem cells are used to generate mice homozygous for the necessary deletions. Step 2: Crossbreeding these mice results in mice homozygous for these deletions, rendering them incapable of producing mouse immunoglobulin. Step 3: Human heavy- and light chain DNA is introduced into a mouse germ line via yeast artificial chromosomes (YACs). The heavy- and light chain YACs are then introduced into mice. Breeding of these mouse strains results in transgenic mice producing both human and mouse antibodies. Step 4: Mice incapable of producing mouse immunoglobulin are crossbred with the transgenic mice (containing both human and mouse antibodies). The resultant strain is the XenoMouse® EDUCATION 43
