ChimericMouseHumanAntibody

The J22.9-xi chimeric mouse/human antibody against human BCMA (PDB ID: 4ZFO) is a chimeric antibody generated against B-cell maturation antigen (BCMA) in Homo sapiens using Mus musculus myeloma cells. The (Fab)2 region, or antigen-binding fragment, of J22.9-xi chimeric antibody is presented in complex with human BCMA, creating a chimeric antibody-antigen complex (1). BCMA, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a protein specific to the surface of plasma cells and mature B cells, particularly in individuals diagnosed with multiple myeloma (2). Multiple myeloma is a blood malignancy that leads to the proliferation and accumulation of diseased plasma cells in the bone marrow and the eventual formation of cancerous masses (2, 3). Individuals suffering from multiple myeloma have a median life expectancy of five to seven years with the aid of existing therapeutic drugs; ultimately, the disease remains incurable. However, therapies using monoclonal antibodies have shown promise. The J22.9-xi chimeric antibody has a high affinity for the ligand binding site of BCMA, blocking the BCMA receptor from binding to its two ligands: a proliferation-inducing ligand (APRIL) and B-cell activating factor (BAFF) (1). When bound to BCMA, APRIL and BAFF promote the survival of normal plasma cells in the bone marrow, but also the proliferation of malignant multiple myeloma plasma cells (4). By blocking the signaling process of BCMA, J22.9-xi chimeric antibody efficiently depletes murine multiple myeloma models of malignant plasma cells, both in vitro and in vivo. The J22.9-xi chimeric antibody is thus a promising new target for treating multiple myeloma, as well as other autoimmune diseases (1).

The structure of J22.9-xi chimeric antibody determines its binding and subsequent blocking of the BCMA receptor, governing the anti-tumor response in murine multiple myeloma models (1). The molecular weight of J22.9-xi is 105919.70 Da and the isoelectric point is 8.47, as calculated by ExPASy (5). The (Fab)2 region of the J22.9-xi chimeric antibody, consisting of four polypeptide chains of Mus musculus origin, is bound to the extracellular (N-terminal) domain of BCMA, which consists of two polypeptide chains of Homo sapiens origin, creating a J22.9-xi:BCMA antibody-antigen complex. Chains B and L make up the light chain of the J22.9-xi chimeric antibody, with 213 and 214 residues, respectively. Chains A and H are identical, making up the heavy chain, with 221 residues each. Human BCMA, specifically TNFR superfamily member 17, is comprised of the two identical chains F and K with 54 residues (1, 6). J22.9-xi chimeric antibody interacts with BCMA primarily through hydrophobic interactions, the majority of which occur at the three complementarity determining regions (CDRs) on the J22.9-xi light chain. The heavy chain also interacts with BCMA through hydrophobic interactions at three CDRs, but only six residues are involved. The J22.9-xi:BCMA interface also involves multiple water molecules, which mediate the region of contact. The interface of the complex occurs at roughly the same BCMA epitope that binds APRIL and BAFF, with J22.9-xi covering 10 of 14 residues bound by APRIL and 10 of 16 residues bound by BAFF. Furthermore, the BCMA epitope has a DXL motif (a repeating Asp-X-Leu sequence) in the central cavity of its antigen binding site, which is completely covered in the J22.9-xi:BCMA complex as well as when bound to the ligands APRIL and BAFF. Thus, the significant overlap of the binding regions on BCMA for APRIL and BAFF with those of J22.9-xi provide evidence for the signal blocking activity of J22.9-xi chimeric antibody (1).

The overall structure of J22.9-xi chimeric antibody is in large part determined by its secondary structure, consisting mostly of beta sheets and alpha helices. The (Fab)2 region of J22.9-xi chimeric antibody is made up of a light chain and a heavy chain, each with three CDRs. Chain A of the heavy chain contains 21 beta strands (52% beta sheet) and seven 3/10-helices (9% helical). The identical chain H of the heavy chain contains 23 beta strands (52% beta sheet) and five 3/10-helices (6% helical). Chain B and chain L of the light chain each contain 21 beta strands (48% beta sheet), one 3/10-helix, and two alpha-helices (5% helical). The extracellular BCMA region bound to the antibody consists of two identical chains (F and K) with two beta strands (14% beta sheet) and two 3/10-helices (12% helical). Furthermore, the J22.9-xi:BCMA complex has two ligands bound to its light chains: BTB and copper (II) ion. BTB, whose structure is shown in Figure 1, binds to chain L at Asp-185 and to chain B at Asp-185 and Lys-188, while copper (II) ion binds to both chain L and B at His-189, and once more to chain L at Asp-1. The binding sites of both ligands are either in or near alpha-helices in the light chains, indicating that they help stabilize the secondary structures at those residues (6). While the predominant beta sheets and few alpha helices, as well as the intermittent random coils, grant J22.9-xi chimeric antibody its overall structure, the primary structure is what determines the binding interactions in the J22.9-xi:BCMA complex.

The interactions between several key residues on J22.9-xi chimeric antibody and BCMA, including hydrophobic interactions and hydrogen bonds, give rise to the antibody-antigen complex at the binding site. J22.9-xi chimeric antibody interacts with BCMA via both its light chain and heavy chain, with the light chain contributing more residues to the binding site (Figure 2). The following residues interact by hydrophobic contacts between each light chain (stated first) and BCMA (stated last): Ser-67 to Thr-32; Leu-96, Gln-89, Ala-34, and Tyr-91 to Leu-17; Tyr-91 to Ser-16; Phe-49 and Phe-55 to Leu-18; Ser-50 and Ser-31 to Arg-27; Leu-53 to Tyr-13; Leu-53 to Leu-26; and Tyr-94 to His-19. There are also several hydrogen bonds between residues on each light chain(stated first) and residues on BCMA (stated last): Tyr-91 and Ser-50 to Asp-15, and Ser-50 to Tyr-13. There are three CDR regions on each light chain, consisting of a total of eleven residues, where four are aromatic hydrophobic (2 Tyr, 2 Phe), three are hydrophobic (Ala, 2 Leu), and four are polar (Gln, 3 Ser). The corresponding binding site on BCMA has only nine interacting residues, including a DXL motif that takes the form of Asp-15, Ser-16, and Leu-17 (1). The DXL motif is significant because it interacts with the binding cavity of J22.9-xi as well as the ligands APRIL and BAFF, meaning that binding to the chimeric antibody will exclude binding of the ligands at the same BCMA epitope (1, 7). The heavy chains of J22.9-xi chimeric antibody have a less extensive contribution to the binding site, yet they still interact with BCMA at three CDR regions each. The following residues interact by hydrophobic contacts between each heavy chain (stated first) and BCMA (stated last): Leu-99, Tyr-100, and Ala-106 to Leu-18; Tyr-101 to Ala-20, Pro-23, and Ile-22; Trp-33 to His-19. There is also one hydrogen bond that forms between Glu-50 on each heavy chain and His-19 on BCMA. The six residues that are part of the CDR regions of each heavy chain consist of three aromatic hydrophobic (2 Tyr, Trp), two hydrophobic (Leu, Ala), and one negatively charged (Glu) amino acid. The corresponding binding site on BCMA has five interacting residues, three of which are hydrophobic (Ala, Leu, Ile), one of which is positively charged (His), and one of which is Pro (1). The residue interactions at the binding site of J22.9-xi chimeric antibody and the BCMA epitope are critical to its function as a multiple myeloma tumor suppressant.

The J22.9-xi chimeric antibody and associated human BCMA have structural similarities to many other proteins and monoclonal antibodies. The Position-Specific Iterated Basic Local Assignment Search Tool (PSI-BLAST) and the Dali server were used to find proteins with structures comparable to the protein of interest, J22.9-xi. PSI-BLAST finds proteins with similar primary structures within a large protein database, assigning an E value to each comparison protein (subject) to denote the degree of sequence homology to the protein of interest (query). The E value is determined by the number of gaps, which are amino acid sequences present in the query but absent in the subject; E values below 0.05 represent significant similarity (8). The Dali server finds proteins with similar tertiary structures by using a sum-of-pairs method, which compares intramolecular distances, assigning a Z score to specify structural similarity. Z scores greater than 2 are significant, indicating similar folds (9). A query in both servers yielded the AT/MEDI4893 Fab (PDB ID: 4U6V) complex, specifically the Fab heavy chain of the monoclonal antibody, as a suitable comparison protein. The AT/MEDI4893 Fab complex had an E value of 4e-123 and a Z score of 27.7, both of which fall within the boundaries for significant structural similarities (8, 9).

The Fab heavy chain of MEDI4893 has significant structural similarities to the heavy chain of J22.9-xi chimeric antibody, containing 21 beta strands (51% beta sheet) and six 3/10 helices (8% helical) in a chain of comparable size (225 residues total). The overall antibody-antigen complexes also have interesting similarities. MEDI4893 is a human monoclonal antibody that binds to alpha-toxin (AT), a virulent toxin produced by Staphylococcus aureus that causes skin and soft tissue infections. Unlike human BCMA, AT is a large heptomeric complex, but its binding site to MEDI4893 is similar to that of J22.9-xi chimeric antibody. AT binds to a pocket between the Fab light and heavy chain regions of the MEDI4893 antibody, albeit with different residue interactions than the J22.9-xi:BCMA complex. MEDI4893 Fab antibody serves a similar therapeutic function in that it binds to AT at the same residues that typically bind to its receptor in humans, effectively blocking the signaling of the toxin that leads to disease (10). By studying the significant similarities and important differences between comparable proteins, like the AT/MEDI4893 Fab complex, further structural and functional details of the J22.9-xi:BCMA complex will be revealed.

J22.9-xi: Chimeric Mouse/Human Antibody Against Human BCMA (PDB ID: 4ZFO) from Homo sapiens and Mus musculus