Natural and man-made V-gene repertoires for antibody discovery

Abstract

Antibodies are the fastest-growing segment of the biologics market. The success of antibody-based drugs resides in their exquisite specificity, high potency, stability, solubility, safety, and relatively inexpensive manufacturing process in comparison with other biologics. We outline here the structural studies and fundamental principles that define how antibodies interact with diverse targets. We also describe the antibody repertoires and affinity maturation mechanisms of humans, mice, and chickens, plus the use of novel single-domain antibodies in camelids and sharks. These species all utilize diverse evolutionary solutions to generate specific and high affinity antibodies and illustrate the plasticity of natural antibody repertoires. In addition, we discuss the multiple variations of man-made antibody repertoires designed and validated in the last two decades, which have served as tools to explore how the size, diversity, and composition of a repertoire impact the antibody discovery process.

Keywords: antibody structure; antigen-binding site; structure-function relationship; therapeutic antibodies.

Figure 1

Ribbon representation of an intact IgG molecule (PDBID: 1IGT). The heavy chains are shown in dark blue, while the Light chains are colored in light blue. The carbohydrate moieties attached to the C_H2 domains are represented with sticks. The figure was produced using PyMol (DeLano, 2002. The PyMOL molecular graphics system. Delano Scientific, San Carlos, CA).

Figure 2

Ribbon representation of a V_H (left) domain and a C_H1 (right) domain. CDRs are colored in yellow (CDR-1), orange (CDR-2), and red (CDR-3). Note the insertion in the V_H domain with respect to the C_H1 domain of two β-strands, C' and C”, and the loop linking them, which contains the CDR-H2. The coordinates used to produce the Figure were the same as in Figure 1. The figure was generated with PyMol.

Figure 3

Ribbon representation of a Fv fragment seen from the antigen perspective. V_H is colored in dark blue, while V_L is colored in light blue. CDRs are colored in yellow (CDR-1), orange (CDR-2), and red (CDR-3). The coordinates used to produce the Figure are the same as in Figure 1. The Figure was generated with PyMol.

Figure 4

Trace representation of 99 unique mid to high resolution (= 3.0 Å) Fv structures, including 30 in complex with proteins, 34 with peptides, and 35 with haptens. V_H colored in dark blue. V_L colored in light blue. Ligands are colored in orange. The coordinates used to generate the Figure are listed in Ragunathan et al. (2012). The structures were superposed in Discovery Studio using the FR Cα atoms.

Figure 5

Connolly (1983) surfaces of representative anti-protein, anti-peptide and anti-hapten Fvs, shown from the antigen perspective. A gradient from red (contact) to white (no contact) represents the SDRUs of antibodies recognizing generic ligands. The surfaces were generated by running a 7.0-Å radius probe over the Fv after removing the CDR-H3 loop (yellow ribbon) to better represent the surface common to all antibodies. Note the variation in size of the surface from anti-protein, a large surface, to anti-hapten antibodies, a small one. On the bottom, a ribbon representation of a Fv in the same orientation as the Fvs displaying the Connolly surface to indicate the position of the CDRs. Also note the protruding CDR-L1 in the anti-peptide and anti-hapten Fvs, which modulates the topography of the antigen-binding site. The ribbon figure was generated with PyMol, while the Connolly surface figures were produced using Discovery Studio.

Figure 6

Ribbon representation of Llama VHH (left) and Shark IgNAR (right) in complex with Hen Egg White Lysozyme (HEL). PDBID 1OP6 was used to represent the VHH:HEL complex. PDBID 2I26 was used to represent the IgNAR:HEL complex. Note the protruding IgNAR CDR-H3 blocking the active site of HEL, which contrasts with the bended VHH CDR-H3 that recognizes a flat epitope. The Figure was generated using PyMOL.

Figure 7

Model of the human anti-peptide (left) and mouse anti-peptide (right) repertoires. On the top, side view of the Connolly surface of the models. Bottom, models seen from the antigen perspective. Invariant V_Ls are colored in brown. V_Hs are colored in gray. Within the antigen-binding site, green represents fully randomized positions, i.e., 20 amino acids; blue positions diversified to Tyr, Asp, Ala, and Ser; red positions diversified to incorporate the most common anti-peptide SDRs. PDBID: 1MCP and 2IGF were used as template for modeling human V_L and V_H, respectively. Coordinates of the antibody 26-10 (PDBID 1IGI) were used for modeling the mouse anti-peptide repertoire. The models and Figures were created in Discovery Studio.