Cary Queen

Genetically engineered deglycosylation of the variable domain increases the affinity of an anti-CD33 monoclonal antibody.

M195 is a murine monoclonal antibody that binds to the CD33 antigen and is being tested for the treatment of myeloid leukemia. Surprisingly, a complementarity determining region (CDR)-grafted, humanized M195 antibody displayed a several-fold higher binding affinity for the CD33 antigen than the original murine antibody. Here we show that the increase in binding affinity resulted from eliminating an N-linked glycosylation site at residue 73 in the heavy chain variable region in the course of humanization. Re-introducing the glycosylation site in the humanized antibody reduces its binding affinity to that of the murine antibody, while removing the glycosylation site from the murine M195 variable domain increases its affinity. The removal of variable region carbohydrates may provide a method for increasing the affinity of certain monoclonal antibodies with diagnostic and therapeutic potential.

The Integrity of the Ball-and-Socket Joint Between V and C Domains Is Essential for Complete Activity of a Humanized Antibody

AF2 is a high affinity murine Ab possessing potent neutralizing activity against human IFN-γ. In carrying out the modifications to humanize this Ab, we discovered that an initial version displayed affinity for IFN-γ that was slightly less than that of AF2, but exhibited IFN-γ-neutralizing activity that was severely diminished. Characterization via site-directed mutagenesis revealed that the majority of this loss in IFN-γ-neutralizing activity was due to altering the VH framework residue at position 11. VH position 11 is distal to the binding surface of the Ab; however, it, along with residues 110 and 112, have been identified as forming the socket of a molecular ball-and-socket joint between the V and C domains of the Ig Fab, which influences the elbow angle between these domains. To determine whether disrupting the structure of this joint was the basis for reduced IFN-γ-neutralizing capacity, we altered residue 148 of CH1, which with residue 149 comprises the corresponding ball portion of the joint. Changing this single CH1 domain residue diminished the ability of the Ab to neutralize IFN-γ to a level similar to that observed with the VH alteration. Thus, an intact ball-and-socket joint between the V and C domains in AF2 is required for potent neutralization of IFN-γ. These results suggest the importance of the elbow angle between Ig V and C domains in Ab activity, and support the hypothesis that this joint can be an important functional element of Ab structure.

Murine and humanized constructs of monoclonal antibody m195 (anti‐cd33) for the therapy of acute myelogenous leukemia

Long‐term survival rates of patients with acute myelogenous leukemia treated with intensive chemotherapy are 15–20%, despite efforts to develop new treatment strategies. Murine M195 (131I‐M195), an anti‐CD33, immunoglobulin (Ig) G2a monoclonal antibody has reactivity restricted to early myeloid cells and myeloid leukemic blasts but not hematopoietic progenitors. Previous trials in patients with relapsed or refractory myeloid leukemia showed that 131I‐M195 rapidly targeted to the bone marrow and internalized into target cells.

Comparison of the three-dimensional structures of a humanized and a chimeric Fab of an anti-gamma-interferon antibody.

The objective of this work is to compare the three‐dimensional structures of “humanized” and mouse–human chimeric forms of a murine monoclonal antibody elicited against human γ‐interferon. It is also to provide structural explanations for the small differences in the affinities and biological interactions of the two molecules for this antigen. Antigen‐binding fragments (Fabs) were produced by papain hydrolysis of the antibodies and crystallized with polyethylene glycol (PEG) 8000 by nearly identical microseeding procedures. Their structures were solved by X‐ray analyses at 2.9 Å resolution, using molecular replacement methods and crystallographic refinement. Comparison of these structures revealed marked similarities in the light (L) chains and near identities of the constant (C) domains of the heavy (H) chains. However, the variable (V) domains of the heavy chains exhibited substantial differences in the conformations of all three complementarity‐determining regions (CDRs), and in their first framework segments (FR1). In FR1 of the humanized VH, the substitution of serine for proline in position 7 allowed the N‐terminal segment (designated strand 4‐1) to be closely juxtaposed to an adjacent strand (4‐2) and form hydrogen bonds typical of an antiparallel β‐pleated sheet. The tightening of the humanized structure was relayed in such a way as to decrease the space available for the last portion of HFR1 and the first part of HCDR1. This compression led to the formation of an α‐helix involving residues 25–32. With fewer steric constraints, the corresponding segment in the chimeric Fab lengthened by at least 1 Å to a random coil which terminated in a single turn of 310 helix. In the humanized Fab, HCDR1, which is sandwiched between HCDR2 and HCDR3, significantly influenced the structures of both regions. HCDR2 was forced into a bent and twisted orientation different from that in the chimeric Fab, both at the crown of the loop (around proline H52a) and at its base. As in HCDR1, the last few residues of HCDR2 in the humanized Fab were compressed into a space‐saving α‐helix, contrasting with a more extended 310 helix in the chimeric form. HCDR3 in the humanized Fab was also adjusted in shape and topography. The observed similarities in the functional binding activities of the two molecules can be rationalized by limited induced fit adjustments in their structures on antigen binding. While not perfect replicas, the two structures are testimonials to the progress in making high affinity monoclonal antibodies safe for human use. Copyright

Abstract 3256: Highly potent monoclonal and bispecific anti-angiogenic antibodies to VEGF and angiopoietin-2

The angiogenic factor Vascular Endothelial Growth Factor (VEGF) is a well-established target for cancer therapy. The humanized monoclonal antibody (mAb) bevacizumab (Avastin®), which binds and neutralizes VEGF, is widely prescribed for the treatment of colon, lung and certain other tumors, but typically provides a relatively modest survival benefit of several months. It is unknown whether the limited benefit is due to redundancy in angiogenic pathways or to the inability of bevacizumab at the doses used to fully neutralize VEGF in the tumor microenvironment. However, the exploration of higher doses of bevacizumab in the clinical setting has been very limited due to lack of incentive to perform dose-ranging studies for an already approved drug, the expense of the drug, and concern about toxicity. To determine whether more effective neutralization of VEGF would be possible and useful, we have therefore used an intensive immunization protocol to generate a new murine mAb designated VE1 that binds VEGF with very high affinity. A humanized form of this mAb, HuVE1, retains the full affinity of VE1 and binds VEGF about 5-fold better than bevacizumab. In side-by-side comparisons, HuVE1 also blocked the binding of VEGF to its receptor with an IC50 that is 5 to 10-fold lower than for bevacizumab, and it correspondingly inhibited VEGF-induced proliferation of HUVEC about 5-fold better than bevacizumab. HuVE1 almost completely blocked growth of primary human hepatocellular carcinoma (HCC) xenografts in one model, and showed a trend toward better efficacy than bevacizumab in a second HCC xenograft model. For potentially even greater anti-angiogenic efficacy, we developed a humanized mAb HuA2T that potently neutralizes angiopoietin-2 (Ang-2), a cytokine that acts through the Tie-2 receptor to promote angiogenesis, especially in combination with VEGF. The synergy between VEGF and Ang-2 provides a strong rationale to develop a bispecific mAb that neutralizes both these factors. We therefore developed such a mAb using the IgG-like bispecific antibody format Bs(scFv)4-IgG consisting of a homodimer of two monomers, each monomer having a single chain Fv from HuVE1 linked to a light chain constant region, and a single chain Fv from HuA2T linked to a heavy chain constant region. While, as is common in construction of bispecific antibodies, this bispecific HuA2T/HuVE1 mAb did lose some binding affinity for VEGF and Ang-2 relative to the original mAbs, its VEGF binding activity was still greater than that of bevacizumab, illustrating the importance of starting with an extremely high activity mAb. Based on its potency in vitro and in animal models, we believe that HuVE1, in either natural or bispecific form, has the potential for greater clinical efficacy than bevacizumab and thus merits further investigation. Citation Format: Hangil Park, April Zhang, Yi Ding, Lihong Wang, Zhengran Wang, Maximiliano Vasquez, Cary Queen, Jin Kim. Highly potent monoclonal and bispecific anti-angiogenic antibodies to VEGF and angiopoietin-2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3256.

Abstract 3493: New, highly potent antibodies to death receptors having Fc mutations to increase antitumor activity

Death receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2 or Apo2) are TNF receptor superfamily members that are receptors for TRAIL (Apo2 ligand), an immunomodulatory cytokine. Binding of TRAIL to DR4 or DR5 can activate the extrinsic apoptotic pathway selectively in tumor cells. Agonist antibodies to death receptors DR4 and DR5 thus have potential for treatment of cancer and may be better than derivatives of TRAIL itself because of their superior pharmacokinetics and receptor selectivity. However, despite dramatic effects in vitro and in preclinical xenograft models, several agonist antibodies to DR4 and especially DR5 have not provided significant therapeutic benefit in clinical trials. While resistance mechanisms in cancer cells are undoubtedly one reason, another factor may be inadequate potency of the antibodies tested. We therefore used intense immunization and screening protocols to develop very potent anti-DR4 and anti-DR5 monoclonal antibodies (mAbs), which were humanized to make the mAbs denoted HuD114 and HuG4.2 respectively. These mAbs were substantially more effective at killing tumor cells in vitro than the mAbs that have been tested in clinical trials. In addition, we introduced one or two mutations into the Fc (constant) regions of these and other potent anti-DR4 and anti-DR5 mAbs in order to increase affinity for the Fc gamma receptor IIb. Cross-linking, as provided by binding of anti-DR4 and anti-DR5 mAbs to Fc gamma receptors on immune cells, is required for effective transmission of an apoptotic signal through the death receptors. Accordingly, introduction of these mutations greatly increased the ability of the mAbs to kill tumor cells in vitro in the presence of human peripheral blood mononuclear cells, and to inhibit the growth of tumor xenografts in mouse models, with two mutations generally more effective than a single mutation. As a complementary approach to increase cross-linking, we also developed bispecific antibodies containing two binding domains from an anti-DR4 mAb and two binding domains from an anti-DR5 mAb. These bispecific mAbs were made in the IgG-like Bs(scFv)4-IgG format consisting of a homodimer of two monomers, each monomer having a single chain Fv from an anti-DR4 mAb linked to a heavy chain constant region, and a single chain Fv from an anti-DR5 mAb linked to a light chain constant region. Such a bispecific mAb was more effective at killing tumor cells than an anti-DR4 or anti-DR5 mAb alone, or even a mixture of anti-DR4 and anti-DR5 mAbs. Based on their potency in vitro and in animal models, we believe that HuD114 and HuG4.2, enhanced by the Fc mutations and/or in bispecific form, have the potential for greater clinical efficacy than previously tested anti-DR4 and anti-DR5 mAbs and thus merit further investigation. Citation Format: Lihong Wang, Yi Ding, Hangil Park, April Zhang, Zhengran Wang, Maximiliano Vasquez, Cary Queen, Jin Kim. New, highly potent antibodies to death receptors having Fc mutations to increase antitumor activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3493.