Abstract CN07-01: Two-in-one antibody: Herceptin variants that target HER2 and VEGF
Authors: Fuh Germaine , Bostrom Jenny 
Published: May 2019

Abstract
Monoclonal antibodies are typically characterized by their exquisite specificity toward single antigens, which has been the basis for the successful use of antibodies as targeted therapeutics. There is, however, increasing appreciation for antibody multi-specificity and how it may play a role in the immune responses. As the total number of antibody producing lymphocytes in each individual is finite (107–9) and the varieties of potential foreign antigens is infinite, the ability of one antibody to recognize more than one antigen may expand the effective size of the antibody repertoire and increase the efficiency of antigen recognition.
To understand the molecular mechanism of how one antibody may accommodate different antigens, previous studies set out to identify unique binding molecules for antibodies distinct from the cognate antigens by selection from synthetic repertoires of peptides or proteins. Although the affinities of these isolated ‘antigens’ are generally weak, they demonstrated that one antigen-binding site is capable of distinct interaction (James et al., 2003; Kramer et al., 1997; Sethi et al., 2006).
We set out to understand the role of antibody multispecificity from a different angle. We want to discover antibodies that are dual specific against two related antigens with high affinity, so as to reveal the mechanism of the multispecific antibodies that are able to play a functional and physiological role due to the high affinity. We also want to identify a general strategy to generate such antibodies against two defined antigens de novo by evolving a monospecific antibody to bind a second antigen. This may provide an option to therapeutically target two molecules with one antibody, and also provide insight into the propensity and mechanism of adding a distinct binding specificity to a matured antibody and the evolution of binding specificity.
We explore whether a mono-specific antibody, HERCEPTIN®, which binds human epidermal growth factor receptor 2 (HER2, ErbB2), can be evolved into one that binds a second binding specificity toward a distinct antigen and maintains its HER2-binding ability. We generated a repertoire of Herceptin antibody variants by mutating in the light chain (LC) complementarity determining regions (CDRs) in a manner that mimic the natural diversity. From this repertoire of Fabs displayed on M13 phage we isolated variants that have 3–17 substitution or insertion compared to Herceptin and can bind the new antigens. Some of them maintained HER2 binding and are thus dual specific. The approach is based on the understanding that modifications of the LC sequence can modulate the binding specificity of antibodies. In addition, many antibodies, with Herceptin as a prime example (Cho et al., 2003; Kelley and O'Connell, 1993), bind the antigens by utilizing mainly the heavy chain (HC) CDRs, suggesting that mutations in the LC CDRs might allow preservation of the original antigen-binding specificity.
We chose to focus on a dual specific variant of Herceptin bH1 that binds vascular endothelial growth factor (VEGF) in addition to HER2 with dissociation constant (Kd) of 300nM and 26 nM, respectively. Further, bH1 exhibited inhibitory activity in the proliferation of VEGF-stimulated endothelial cells (HUVEC) and HER2 overexpressing BT474 breast cancer cell line, suggesting that bH1 retained the HER2-binding function of Herceptin, and acquired a VEGF-blocking activity.
To understand the molecular mechanism of the dual specificity, we solved the crystal structures of bH1 in complex with VEGF (the receptor binding domain, residue 8–109) and HER2 (residue 1–624) at 2.6 anf 2.9 Å resolution. The structure of the bH1/HER2 binding domains (bH1 Fv and domain IV of HER2) superimposes onto the Herceptin/HER2 complex with an r.m.s.d of 0.8 Å, showing that the Herceptin binding epitope on HER2 is retained. The structure of bH1/VEGF complex show that the Fab recognizes an epitope that overlaps with the binding sites of the VEGF receptors and other VEGF binding. Consistent with this observation, bH1 inhibits the VEGF-stimulated growth of endothelial cells.
Comparison of the bH1/VEGF and the bH1/HER2 complexes shows an extensive overlap of the VEGF and HER2 binding sites on bH1; eleven out of the thirteen bH1 residues that are in close contact with HER2 also make contact with VEGF. In the HER2 complex, the LC and HC CDRs contribute approximately equal antigen contact area (53% and 47% respectively) while in the VEGF complex, the LC CDRs constitute nearly 70% of the buried surface.
With exception of CDR-L1, the CDR conformations of the bH1 Fab bound to VEGF are strikingly similar to those observed in bH1 or Herceptin bound to HER2. The capability of CDR-L1 to rearrange appears to be necessary for the dual specificity of bH1.
Shotgun scanning mutagenesis studies were performed to assess the energetic contributions of individual CDR residues to VEGF and HER2 binding and to determine the strategies to improve the two binding affinities. In contrast to the extensive overlap between buried surfaces in the interfaces of bH1 bound to either VEGF or HER2, the two functional paratopes show only limited overlap. The VEGF binding interaction is energetically mediated by the LC CDRs primarily whereas HER2-binding is dominated by HC CDRs. Compared to Herceptin, bH1 maintains the same core hot spot residues for HER2 binding.
The structural and functional studies show that the interactions between bH1 and the two entirely unrelated large proteins are distinct, and are characterized by conformational adaptation of the antigen binding site and differential engagement of VL and VH. The molecular versatility observed in the dual specific bH1 is reminiscent of other antibodies binding distinct molecules.
To facilitate the investigation of the in vivo activity of bH1, we generated versions of bH1 with high affinity and specificity for HER2 and VEGF. We use the information of the shotgun mutagenesis scanning to design two sets of libraries of bH1 variants. From the library based on bH1, we derived bH1–81 (KD=58/6 nM VEGF/HER2). Based on bH1–81, we generated randomize libraries to derive the high affinity bH1–44 (KD= 3/0.2nM VEGF/HER2). The affinity improved bH1 variants inhibit VEGF-induced proliferation of human umbilical vein endothelial cells (HUVEC) and the growth of the HER2 overexpressing breast cancer cell line BT474 with improved potencies correlated with their relative affinities. The high affinity bH1–44 now inhibits the growth of HUVEC and BT474 cells with potencies similar to bevacizumab or Herceptin, respectively.
To examine the two unique activities of bH1–44 in vivo, we employed mouse xenograft tumor models known to be responsive to treatment by anti-VEGF antibodies (Colo205, a human colorectal cancer cell line) or Herceptin (BT474M1, human breast cancer cell line). The bH1–44 treated groups were compared with those treated with anti-VEGF (B20-4.1), Herceptin, or the combination (Herceptin + anti-VEGF). bH1–44 as well as B20-4.1 cross binds murine VEGF, The results of the Colo205 model demonstrate that the VEGF binding component of bH1–44 is inhibiting tumor growth, as Herceptin alone has no effect. The BT474M1 model indicates that the HER2 binding component of bH1–44 is active, since the effect observed with the anti-VEGF control antibody is not significant.
Hence, bH1–44 has demonstrated the pharmacological activity of a VEGF blocking antibody and a Herceptin-like antibody. However, it is not yet known whether bH1–44 is indeed as effective as Herceptin or anti-VEGF administered separately or in combination. The advantage of co-targeting the tumor cell proliferation mediated by HER2 and tumor angiogenesis mediated by VEGF is currently being evaluated in clinical trials combining Herceptin and bevacizumab in breast cancer patients. These studies may indicate the potential of developing dual specific antibodies like bH1–44 as therapeutics. Herein, we demonstrate a general strategy to derived dual specific antibodies that can be applied to create dual specific antibodies against two defined antigens. In contrast to currently reported bispecific formats, which assemble two antigen binding units into one molecule, the dual specific “two-in-one” antibody we describe has the molecular structure of a regular IgG (or Fab). It has all the favourable attributes of an IgG for therapeutic development, such as predictable pharmacokinetic properties, well established manufacturing protocols, choice of Fc-mediated effector functions and bi- or mono-valencies.
We also demonstrated that an antigen-binding site is capable of interacting with two unrelated protein antigens with high affinity. Mutational analysis of bH1 and bH1–44 allows us to derive monospecific HER2 or VEGF antibodies from bH1–44 by two alanine mutations in the LC or HC CDRs, respectively. This further highlights how a limited number of mutations in the antigen-binding site can alter specificity, or add an additional specificity. During development of the natural antibody repertoire, the antigen-binding sites often undergo diversification by exchanging the VL that pairs with a VH. Somatic mutations also occur frequently, in particular among the residues in the periphery of the antigen-binding site. Our studies reveal a mechanism by which one antibody can diverge into many antibodies with distinct specificity profiles, including dual specificity. This mechanism may contribute to the large capacity of the natural antibody repertoire for diverse antigen recognition.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):CN07-01.