Haifan Zhang

Tailoring in Vitro Selection for a Picomolar Affinity Human Antibody Directed against Vascular Endothelial Growth Factor Receptor 2 for Enhanced Neutralizing Activity

Vascular endothelial growth factor (VEGF) and its receptors have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We previously identified several fully human neutralizing anti-VEGF receptor 2 (or kinase inserting domain-containing receptor (KDR)) antibodies from a large antibody phage display library. These antibodies bind specifically to KDR, block VEGF/KDR interaction, and inhibit VEGF-induced proliferation of human endothelial cells and migration of KDR+ leukemia cells. Three of these antibodies, interestingly, share an identical heavy chain variable (VH) sequence. In this report, we constructed a new library comprising the single VH paired with the variable light chain (VL) repertoire obtained from the original naïve human library. Initial in vitro selection revealed that the single VH could pair with a number of different VL while retaining its specificity for KDR. However, a consensus VH/VL pair, clone 1121, was identified after three or four rounds of selection by tailoring the stringency of the panning conditions. Clone 1121 showed a >30-fold higher binding affinity to KDR (Kd, 100 pm) because of improvement on both association and dissociation constants and blocked VEGF/KDR interaction with an IC50 of ∼1 nm, compared with that of 3–4 nm for the parent Fab fragments. Further, clone 1121 was more potent in inhibiting VEGF-stimulated KDR phosphorylation in endothelial cells. A binding epitope mapping study on clone 1121 and one of the parent clones, 2C6, demonstrated that both antibodies interacted with the third immunoglobulin domain within the extracellular region of KDR. Several peptide phage display libraries were utilized to further examine the fine binding specificities of the two antibodies. All of the 2C6-binding peptides are cysteine-constrained, whereas clone 1121 binds to both cysteine-constrained and linear peptides. It is noteworthy that most of the 2C6-binding peptides also cross-react with clone 1121, but none of the clone 1121-specific peptides binds to 2C6, indicating that clone 1121 retained part of the original binding epitope(s) of 2C6 while gaining new binding specificity. Taken together, our observation suggests that clone 1121 may have great clinical potential in anti-angiogenesis therapy. It further underscores the efforts to identify antibodies of high affinity for enhanced biological activities.

Simultaneous Blockade of Both the Epidermal Growth Factor Receptor and the Insulin-like Growth Factor Receptor Signaling Pathways in Cancer Cells with a Fully Human Recombinant Bispecific Antibody

Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR) have been implicated in the tumorigenesis of a variety of human cancers. Effective tumor inhibition has been achieved both experimentally and clinically with a number of strategies that antagonize either receptor activity. Here we constructed and produced two fully human recombinant bispecific antibodies (BsAb) that target both EGFR and IGFR, using two neutralizing human antibodies originally isolated from a phage display library. The BsAb not only retained the antigen binding capacity of each of the parent antibodies, but also were capable of binding to both targets simultaneously as demonstrated by a cross-linking enzyme-linked immunosorbent assay. Furthermore, the BsAb effectively blocked both ligands, EGF and IGF, from binding to their respective receptors, and inhibited tumor cell proliferation as potently as a combination of both the parent antibodies. More importantly, the BsAb were able to completely block activation of several major signal transduction molecules, including Akt and p44/p42 MAP kinases, by both EGF and IGF, whereas each individual parent antibody was only effective in inhibiting those signal molecules activated by the relevant single growth factor. The BsAb molecules retained good antigen binding activity after incubation with mouse serum at 37 °C for up to 6 days. Taken together, our results underscore the benefits of simultaneous targeting multiple growth factor receptor pathways for more efficacious cancer treatment. This report describes the first time use of a recombinant BsAb for targeting two tumor-associated molecules on either a single or adjacent tumor cells for enhanced antitumor activity.

Selection of high affinity human neutralizing antibodies to VEGFR2 from a large antibody phage display library for antiangiogenesis therapy

Compelling evidence suggests that vascular endothelial growth factor (VEGF) and its receptors play an important role in angiogenesis associated with tumor growth and metastasis. VEGF exerts its biologic activities through 2 transmembrane tyrosine kinase receptors: the fms‐like tyrosine kinase receptor (Flt‐1, or VEGFR1) and kinase insert domain‐containing receptor (KDR or VEGFR2). We have previously produced a panel of antibodies directed against KDR from mice immunized with the recombinant form receptor. These antibodies efficiently neutralized VEGF‐induced KDR activation and mitogenesis of human umbilical vascular endothelial cells (HUVEC). Murine antibodies, however, may not be suitable candidates for human therapy because of their propensity to elicit human anti‐mouse antibody response. Here we isolated several high‐affinity human Fab antibody fragments directed against KDR from an antibody phage display library constructed from the pooled B lymphocytes of nonimmunized healthy human donors. These human Fab fragments bind specifically to KDR with nanomolar affinity and block KDR/VEGF interaction with IC50 of approximately 2–20 nM. Further, they effectively inhibit VEGF‐stimulated mitogenesis of HUVEC and migration of human leukemia cells. Epitope mapping studies demonstrated that all neutralizing human antibodies bound the epitope(s) located within the first 3 N‐terminal immunoglobulin‐like domains of KDR, the same region that encompasses the binding site of VEGF. Our results suggest that these human anti‐KDR antibodies may have potential application in the treatment of cancer and other diseases in which pathologic angiogenesis occurs.

Fab-scFv fusion protein: an efficient approach to production of bispecific antibody fragments.

The clinical development of bispecific antibodies (BsAb) as therapeutics has been hampered by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods. Here, we describe an efficient approach for the production of a novel bispecific antibody fragment by genetically fusing a single-chain Fv (scFv) to the C-terminus of either the light chain or the heavy chain of a Fab fragment of different antigen-binding specificity. The bispecific Fab-scFv fragments were expressed in a single Escherichia coli host and purified to homogeneity by a one-step affinity chromatography. Two different versions of the bispecific Fab-scFv fragments were constructed using two antibodies directed against the two tyrosine kinase receptors of vascular endothelial growth factor. These bispecific antibody fragments not only retained the antigen-binding capacity of each of the parent antibodies, but also are capable of binding to both targets simultaneously as demonstrated by a cross-linking ELISA. Further, the bispecific antibodies were comparable to their parent antibodies in their potency in blocking ligand binding to the receptors and in inhibiting ligand-induced biological activities. This design for BsAb fragments should be applicable to any pair of antigen specificities.

Single Variable Domain-IgG Fusion A NOVEL RECOMBINANT APPROACH TO Fc DOMAIN-CONTAINING BISPECIFIC ANTIBODIES

Both laboratory and early clinical studies to date have demonstrated that bispecific antibodies (BsAb) may have potentially significant application in cancer therapy. The clinical development of BsAb as therapeutics has been hampered, however, by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods. In recent years, a variety of recombinant methods has been developed for efficient production of BsAb, both as antibody fragments and as full-length IgG-like molecules. Here we describe a novel recombinant approach for the production of an Fc domain-containing, IgG-like tetravalent BsAb, with two antigen-binding sites to each of its target antigens, by genetically fusing a single variable domain antibody to the N terminus of the light chain of a functional IgG antibody of different specificity. A model BsAb was constructed using a single variable domain antibody to mouse platelet-derived growth factor receptor α and a conventional IgG antibody to mouse vascular endothelial growth factor receptor 2. The BsAb was expressed in mammalian cells and purified to homogeneity by one-step protein A affinity chromatography. Furthermore, the BsAb retains the antigen binding specificity and the receptor neutralizing activity of both of its parent antibodies. This design and expression of Fc domain-containing, IgG-like BsAb should be applicable to the construction of similar BsAb from antibodies recognizing any pair of antigens.

Abstract C173: Anti-PD-L1 antibody-based IL-15 immunocytokine has enhanced antitumor immunity

Immune checkpoint antagonists to PD-1/PD-L1 and immunostimulating cytokines such as IL-15 have shown success to some extent in certain clinical settings across multiple cancer types. However, the full potential of the checkpoint inhibitor is limited due to impaired overall antitumor immunity, and cytokine as single agent has insufficient half-life and systemic toxicities due to the lack of target specificity. To overcome these challenging hurdles, we developed a bifunctional fusion protein, KD-033, composed of an antibody specific for PD-L1 and complex of IL-15Rα sushi domain/IL-15 as a novel immunotherapeutic agent for achieving better antitumor efficacy. Previously, we presented the generation and characteristics of a prototype of bifunctional fusion protein and its potential of in vivo antitumor activity. Here, we report a genetically modified fusion protein that has enhanced immunological activity and capability to achieve stronger antitumor efficacy in tumor models in comparison with either single agent. Our data indicate that the improved bifunctional fusion protein has favorable thermal stability and can be efficiently expressed in mammalian cells. The bifunctional fusion protein has higher affinity to PD-L1, silenced binding activity to Fc receptors and better ability to increase the secretion of Th1 cytokine, i.e. gamma IFN and the cytotoxicity of CD8 T-cells and NK cells to tumor cells as assessed in immunological assays. In preclinical study, KD033 had stronger anti-tumor efficacy in controlling primary tumor growth and prolonging the survival of tumor bearing mice in a number of mouse tumor models including those aggressive tumor models. Furthermore, the PD-L1 targeted bifunctional protein had significantly less cytokine-related toxicity when compared to non-targeted full IgG/IL-15Rα sushi domain/IL-15 fusion protein in vivo. These results demonstrate that KD033 has the capacity of targeting IL-15-stimulated innate and adaptive immune effectors into local tumor sites, thereby effectively controlling tumor progression while having minimized potential adverse effect in vivo. The preclinical studies of the novel immunotherapeutics warrant further investigation towards the clinical development of the bifunctional immunotherapeutic agent for cancer treatment. Citation Format: Yan Wu, Zhaojing Zhong, Stella Martomo, Dan Lu, Zhanna Polonskaya, Xenia Luna, Haifan Zhang, Zhikai Zhang, Zhun Wang, Leo Liu, Jeegar Patel, James Tonra, Henry Li, Larry Witte, Sam Waksal, Zhenping Zhu. Anti-PD-L1 antibody-based IL-15 immunocytokine has enhanced antitumor immunity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C173.

Abstract 572: A novel anti-PDL1 x anti-VEGFR2 bispecific antibody for enhanced antitumor immunity

The importance of VEGF/VEGFR2 signaling in cancer growth and metastasis has been clearly highlighted by the therapeutic benefits of bevacizumab (Avastin), a humanized antibody to VEGF, and Cyramza, a fully human antibody to VEGFR2, in multiple cancer treatment modalities. Recently, immune checkpoint antagonistic antibodies to PD1 and PDL1 have shown some success in a variety of clinical settings across multiple cancer types. The overall clinical efficacy of these individual antibody therapies has been, however, rather limited and, only evident in a fraction of patients. To this end, combinations of antibodies against both VEGF/VEGFR2 and PD1/PDL1 may represent promising approaches to further enhance the antitumor efficacy of individual antibody therapies. In this study, we engineered a bispecific anti-PDL1 x anti-VEGFR2 antibody using two fully human antibodies derived from antibody phage display libraries. In this bispecific format, a high affinity single chain antibody to PDL1 was genetically fused to the C-terminus of the heavy chain of a conventional IgG antibody against VEGFR2. The bispecific antibody was efficiently expressed in mammalian cells and could be purified to homogeneity via single step affinity chromatography. The bispecific antibody retained the binding activity of its parental antibodies to PDL1 and VEGFR2, and strongly blocked both VEGF/VEGFR2 and PDL1/PD1 interaction. Further, the bispecific antibody inhibited VEGF-stimulated VEGFR2 phosphorylation and proliferation of endothelial cells, and promoted proliferation of human T cells and secretion of cytokine such as IL2 and INFγ. The bispecific antibody is currently being evaluated in vivo in relevant animal models. Citation Format: Dan Lu, Zhanna Polonskaya, Haifan Zhang, Xenia Luna, Stella Martomo, Zhikai Zhang, Zhaojing Zhong, Yan Wu, Jeegar Patel, James Tonra, Larry Witte, Sam Waksal, Zhenping Zhu. A novel anti-PDL1 x anti-VEGFR2 bispecific antibody for enhanced antitumor immunity. [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 572.

Abstract 4997: Novel anti-PD-L1/IL-15 bifunctional immunotherapeutics potentiates antitumor immunity

Target-based immunocytokine approaches have been reported to be efficacious in the control of tumor growth in preclinical and clinical studies. By targeting cytokines-activated immune effectors to local tumor sites, an antibody-based immunocytokine is able to achieve antitumor immunity in the tumor microenvironment while reducing cytokines-mediated systemic side effects. Recently, immune checkpoint antagonists to PD-1/PD-L1 have shown success in certain clinical settings across multiple cancer types. However, the full potential of the checkpoint inhibitor is limited due to impaired overall antitumor immunity. It is therefore desirable to develop immunotherapeutics with the capacity of simultaneously inhibiting immunosuppressive pathways and stimulating immune effector cells to potentiate innate and adaptive immune responses against tumor growth. To this end, we generated a bifunctional fusion protein, KD033, composed of an antibody specific for PD-L1 and IL-15 as a novel immunocytokine for achieving better immunotherapeutic efficacy against tumors. Previously, we demonstrated that KD033 has an enhanced immunological activity and stronger antitumor efficacy in some syngeneic mouse tumor models in comparison to single agents. In the present report, we show that the mechanisms of actions of the bifunctional protein in the enhancement of antitumor immune responses results from an increase in Th1 cytokine secretion, the expansion and cytotoxicity of CD8 T-cells and NK cells and a decrease in immunosuppressive cells, i.e. regulatory T cells and myeloid derived suppressive cells in a number of preclinical experimental models. In the preclinical studies, KD033 regimens, with the unique immunological properties, led to stronger anti-tumor efficacy in controlling primary tumor growth and prolonging the survival of tumor bearing mice in a number of mouse tumor models including PDX and GEMM tumor models. Importantly, the PD-L1-targeted IL-15 bifunctional protein had significantly less cytokine-related toxicity when compared to non-targeted full IgG antibody-IL-15 fusion protein in vivo. These results further elucidate the capacity of targeting IL-15-stimulated innate and adaptive immune effector cells into tumor microenvironment, thereby effectively controlling tumor progression while having minimized adverse effect in vivo. These encouraging preclinical results of the novel immunotherapeutics suggest further advancement of this innovative therapeutic candidate towards clinical development for cancer treatment. Citation Format: Yan Wu, Zhaojing Zhong, Stella Martomo, Dan Lu, Haifan Zhang, Zhanna Polonskaya, Xenia Luna, Zhikai Zhang, Zhun Wang, Leo Liu, Jeegar Patel, James Tonra, Henry Li, Larry Witte, Sam Waksal, Zhenping Zhu. Novel anti-PD-L1/IL-15 bifunctional immunotherapeutics potentiates antitumor immunity. [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 4997.