Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

We developed an IgG1 domain-tethering approach to guide the correct assembly of 2 light and 2 heavy chains, derived from 2 different antibodies, to form bispecific monovalent antibodies in IgG1 format. We show here that assembling 2 different light and heavy chains by sequentially connecting them with protease-cleavable polypeptide linkers results in the generation of monovalent bispecific antibodies that have IgG1 sequence, structure and functional properties. This approach was used to generate a bispecific monovalent antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor that: 1) can be produced and purified using standard IgG1 techniques; 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies.     

Human monomeric antibody fragments to TRAIL-R1 and TRAIL-R2 that display potent in vitro agonism

Apoptosis through the TRAIL receptor pathway can be induced via agonistic IgG to either TRAIL-R1 or TRAIL-R2. Here we describe the use of phage display to isolate a substantive panel of fully human anti-TRAIL receptor single chain Fv fragments (scFvs); 234 and 269 different scFvs specific for TRAIL-R1 and TRAIL-R2 respectively. In addition, 134 different scFvs that were cross-reactive for both receptors were isolated. To facilitate screening of all 637 scFvs for potential agonistic activity in vitro, a novel high-throughput surrogate apoptosis assay was developed. Ten TRAIL-R1 specific scFv and 6 TRAIL-R2 specific scFv were shown to inhibit growth of tumor cells in vitro in the absence of any cross-linking agents. These scFv were all highly specific for either TRAIL-R1 or TRAIL-R2, potently inhibited tumor cell proliferation, and were antagonists of TRAIL binding. Moreover, further characterization of TRAIL-R1 agonistic scFv demonstrated significant anti-tumor activity when expressed and purified as a monomeric Fab fragment. Thus, scFv and Fab fragments, in addition to whole IgG, can be agonistic and induce tumor cell death through specific binding to either TRAIL-R1 or TRAIL-R2. These potent agonistic scFv were all isolated directly from the starting phage antibody library and demonstrated significant tumor cell killing properties without any requirement for affinity maturation. Some of these selected scFv have been converted to IgG format and are being studied extensively in clinical trials to investigate their potential utility as human monoclonal antibody therapeutics for the treatment of human cancer.     

Chronic lymphocytic leukemic cells exhibit apoptotic signaling via TRAIL-R1

Clinical trials have been initiated with Apo2L/TRAIL (Genentech) and agonistic mAbs to TRAIL receptors, -R1 and -R2 (Human Genome Sciences). The apoptosis-inducing ability of these mAbs and different TRAIL preparations, in the presence or absence of histone deacetylase inhibitors (HDACi), varied markedly against primary chronic lymphocytic leukaemia (CLL) cells and various tumor cell lines, demonstrating an unanticipated preferential apoptotic signaling via either TRAIL-R1 or -R2. Contrary to literature reports that TRAIL-induced apoptosis occurs primarily via signaling through TRAIL-R2, CLL cells, in the presence of HDACi, undergo predominantly TRAIL-R1-mediated apoptosis. Consequently, Apo2L/TRAIL, which signals primarily through TRAIL-R2, is virtually devoid of activity against CLL cells. To maximize therapeutic benefit, it is essential to ascertain whether a primary tumor signals via TRAIL-R1/-R2, prior to initiating therapy. Thus combination of an agonistic TRAIL-R1 Ab and an HDACi, such as the anticonvulsant sodium valproate, could be of value in treating CLL.     

Engineering multivalent antibodies to target heregulin-induced HER3 signaling in breast cancer cells

The use of antibodies in therapy and diagnosis has undergone an unprecedented expansion during the past two decades. This is due in part to innovations in antibody engineering that now offer opportunities for the production of “second generation” antibodies with multiple specificities or altered valencies. The targeting of individual components of the human epidermal growth factor receptor (HER)3-PI3K signaling axis, including the preferred heterodimerization partner HER2, is known to have limited anti-tumor effects. The efficacy of antibodies or small molecule tyrosine kinase inhibitors (TKIs) in targeting this axis is further reduced by the presence of the HER3 ligand, heregulin. To address these shortcomings, we performed a comparative analysis of two distinct approaches toward reducing the proliferation and signaling in HER2 overexpressing tumor cells in the presence of heregulin. These strategies both involve the use of engineered antibodies in combination with the epidermal growth factor receptor (EGFR)/HER2 specific TKI, lapatinib. In the first approach, we generated a bispecific anti-HER2/HER3 antibody that, in the presence of lapatinib, is designed to sequester HER3 into inactive HER2-HER3 dimers that restrain HER3 interactions with other possible dimerization partners. The second approach involves the use of a tetravalent anti-HER3 antibody with the goal of inducing efficient HER3 internalization and degradation. In combination with lapatinib, we demonstrate that although the multivalent HER3 antibody is more effective than its bivalent counterpart in reducing heregulin-mediated signaling and growth, the bispecific HER2/HER3 antibody has increased inhibitory activity. Collectively, these observations provide support for the therapeutic use of bispecifics in combination with TKIs to recruit HER3 into complexes that are functionally inert.     

Selective inhibition of FLICE-like inhibitory protein expression with small interfering RNA oligonucleotides is sufficient to sensitize tumor cells for TRAIL-induced apoptosis

BACKGROUND: Most tumors express death receptors and their activation represents a potential selective approach in cancer treatment. The most promising candidate for tumor selective death receptor-activation is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L, which activates the death receptors TRAIL-R1 and TRAIL-R2, and induces apoptosis preferentially in tumor cells but not in normal tissues. However, many cancer cells are not or only moderately sensitive towards TRAIL and require cotreatment with irradiation or chemotherapy to yield a therapeutically reasonable apoptotic response. Because chemotherapy can have a broad range of unwanted side effects, more specific means for sensitizing tumor cells for TRAIL are desirable. The expression of the cellular FLICE-like inhibitory protein (cFLIP) is regarded as a major cause of TRAIL resistance. We therefore analyzed the usefulness of targeting FLIP to sensitize tumor cells for TRAIL-induced apoptosis. MATERIALS AND METHODS: To selectively interfere with expression of cFLIP short double-stranded RNA oligonucleotides (small interfering RNAs [siRNAs]) were introduced in the human cell lines SV80 and KB by electroporation. Effects of siRNA on FLIP expression were analyzed by Western blotting and RNase protection assay and correlated with TRAIL sensitivity upon stimulation with recombinant soluble TRAIL and TRAIL-R1- and TRAIL-R2-specific agonistic antibodies. RESULTS: FLIP expression can be inhibited by RNA interference using siRNAs, evident from reduced levels of FLIP-mRNA and FLIP protein. Inhibition of cFLIP expression sensitizes cells for apoptosis induction by TRAIL and other death ligands. In accordance with the presumed function of FLIP as an inhibitor of death receptor-induced caspase-8 activation, down-regulation of FLIP by siRNAs enhanced TRAIL-induced caspase-8 activation. CONCLUSION: Inhibition of FLIP expression was sufficient to sensitize tumor cells for TRAIL-induced apoptosis. The combination of TRAIL and FLIP-targeting siRNA could therefore be a useful strategy to attack cancer cells, which are resistant to TRAIL alone.     

Post-translational modification of TRAIL receptor type 1 on various tumor cells and the susceptibility of tumors to TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-R1 and TRAIL-R2) are promising targets for tumor therapy. However, their clinical use is limited because some tumors show resistance to TRAIL-treatment. Here, we analyzed epitopes of nine TRAIL-R1-specific human monoclonal antibodies and demonstrated at least five tentative epitopes on human TRAIL-R1. We found that some of the five were post-translationally modified on some tumor cell lines. Interestingly, one of them, an epitope of TR1-272 antibody (TR1-272-epitope) disappeared on the tumor cells that are more susceptible to TRAIL-induced apoptosis compared to TR1-272-epitope positive cells. Treatment of TR1-272-epitope negative cells with TRAIL induced large cluster formation of TRAIL-R1, while treatment of TR1-272-epiope positive cells with TRAIL did not. These results suggest that TR1-272-antibody might distinguish the TRAIL-R1 conformation that could deliver stronger death signals. Further analysis of epitope-appearance and sensitivity to TRAIL should clarify the mechanisms of TRAIL-induced apoptosis of tumor cells and would provide useful information about tumor therapy using TRAIL and TRAIL-R signaling.     

基因工程双特异抗体研究进展

双特异抗体特别是双特异性单链抗体、亮氨酸接链双特异性抗体、双特异性单链抗体毒素等是近几年来发展起来的很有前途的监床诊疗生物制剂 ,本文对其基因构建、边接肽设计、表达产物及活性等方面的新进展作了重点论述。     

Engineering multivalent antibodies to target heregulin-induced HER3 signaling in breast cancer cells

The use of antibodies in therapy and diagnosis has undergone an unprecedented expansion during the past two decades. This is due in part to innovations in antibody engineering that now offer opportunities for the production of “second generation” antibodies with multiple specificities or altered valencies. The targeting of individual components of the human epidermal growth factor receptor (HER)3-PI3K signaling axis, including the preferred heterodimerization partner HER2, is known to have limited anti-tumor effects. The efficacy of antibodies or small molecule tyrosine kinase inhibitors (TKIs) in targeting this axis is further reduced by the presence of the HER3 ligand, heregulin. To address these shortcomings, we performed a comparative analysis of two distinct approaches toward reducing the proliferation and signaling in HER2 overexpressing tumor cells in the presence of heregulin. These strategies both involve the use of engineered antibodies in combination with the epidermal growth factor receptor (EGFR)/HER2 specific TKI, lapatinib. In the first approach, we generated a bispecific anti-HER2/HER3 antibody that, in the presence of lapatinib, is designed to sequester HER3 into inactive HER2-HER3 dimers that restrain HER3 interactions with other possible dimerization partners. The second approach involves the use of a tetravalent anti-HER3 antibody with the goal of inducing efficient HER3 internalization and degradation. In combination with lapatinib, we demonstrate that although the multivalent HER3 antibody is more effective than its bivalent counterpart in reducing heregulin-mediated signaling and growth, the bispecific HER2/HER3 antibody has increased inhibitory activity. Collectively, these observations provide support for the therapeutic use of bispecifics in combination with TKIs to recruit HER3 into complexes that are functionally inert.     

Potentiation of delayed-type hypersensitivity response to syngeneic tumors in mice prevaccinated with cells modified by hydrostatic pressure and crosslinking

Summary Targeting of immune cells by bispecific antibodies has proven a powerful tool for the investigation of cellular cytotoxicity, lymphocyte activation and induction of cytokine production, as well as to represent an innovative form of immunotherapy for the treatment of cancer. The hallmark of this approach is the use of the specificity of monoclonal antibodies to join target and immune cells by virtue of the dual specificity of bispecific antibodies for the two entities. More precisely the bispecific antibody has two different binding sites, which are capable of recognizing tumor associated antigens on the one hand and lymphocyte activation sites on the other. This process of crosslinking results in the activation of the lymphocyte and triggering of its lytic machinery, as well as lymphokine production. A major advantage of this therapeutic modality is, that use is made of the normal cellular immune defence system and therefore is only associated with minor toxicity. The distinct lymphocyte populations, which can be used for adoptive immunotherapy and the various bispecific antibody preparations, as well as the chimeric immunoglobulin/T cell receptor construction are the major topics of this review.     

A bispecific antibody targeting CD47 and CD20 selectively binds and eliminates dual antigen expressing lymphoma cells

Agents that block the anti-phagocytic signal CD47 can synergize with pro-phagocytic anti-tumor antigen antibodies to potently eliminate tumors. While CD47 is overexpressed on cancer cells, its expression in many normal tissues may create an ‘antigen sink’ that could minimize the therapeutic efficacy of CD47 blocking agents. Here, we report development of bispecific antibodies (BsAbs) that co-target CD47 and CD20, a therapeutic target for non-Hodgkin lymphoma (NHL), that have reduced affinity for CD47 relative to the parental antibody, but retain strong binding to CD20. These characteristics facilitate selective binding of BsAbs to tumor cells, leading to phagocytosis. Treatment of human NHL-engrafted mice with BsAbs reduced lymphoma burden and extended survival while recapitulating the synergistic efficacy of anti-CD47 and anti-CD20 combination therapy. These findings serve as proof of principle for BsAb targeting of CD47 with tumor-associated antigens as a viable strategy to induce selective phagocytosis of tumor cells and recapitulate the synergy of combination antibody therapy. This approach may be broadly applied to cancer to add a CD47 blocking component to existing antibody therapies.     

Bispecific-monoclonal-antibody-directed lysis of ovarian carcinoma cells by activated human T lymphocytes

Summary Two different bispecific hybrid antibodies were established by fusing a hybridoma producing monoclonal antibody (mAb) against the pancarcinoma antigen KS1/4 with either of the two hybridomas OKT3 and 9.3, secreting antibodies reactive with the T cell determinants CD3 and CD28, respectively. The KS1/4 antibody reacts with a 40-kDa cell-surface glycoprotein antigen that is expressed on the surface of a variety of adenocarcinoma cells, including ovarian carcinoma. The ability of the bispecific antibodies 9.3KS1/4 and OKT3KS1/4 to direct peripheral blood mononuclear cells (PBMC) specifically against OVCAR-3 ovarian carcinoma target cells was measured in a 4-h⁵¹Cr-release assay. The bispecific antibodies were four to six times more potent in killing the OVCAR-3 target cells when compared to their parental antibodies either alone or in combination. A dose-dependent response was observed in the 10–10000 ng/ml range. The specificity of the targeting was demonstrated by the complete inhibition of cytotoxic activity following pre-incubation of tumor target cells with the parental mAb and by the lack of killing of KS1/4-negative target cell lines. An evaluation of the efficacy of PBMC from ovarian cancer patients as effector cells revealed that their specific cytotoxicity against OVCAR-3 cells was enhanced severalfold by bispecific antibodies as compared to parental antibodies. Furthermore, stimulation of PBMC with immobilized CD3 and interleukin-2 for 4 days resulted in an enhanced directed killing of human ovarian carcinoma cells by human T effector cells and the bispecific antibodies.     

Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTβR

Bispecific antibodies (BsAbs) represent an emerging class of biologics that achieve dual targeting with a single agent. Recombinant DNA technologies have facilitated a variety of creative bispecific designs with many promising therapeutic applications; however, practical methods for producing high quality BsAbs that have good product stability, long serum half-life, straightforward purification, and scalable production have largely been limiting. Here we describe a protein-engineering approach for producing stable, scalable tetravalent IgG-like BsAbs. The stability-engineered IgG-like BsAb was envisioned to target and crosslink two TNF family member receptors, TRAIL-R2 (TNF-Related Apoptosis Inducing Ligand Receptor-2) and LTβR (Lymphotoxin-beta Receptor), expressed on the surface of epithelial tumor cells with the goal of triggering an enhanced anti-tumor effect. Our IgG-like BsAbs consists of a stability-engineered anti-LTβR single chain Fv (scFv) genetically fused to either the N- or C-terminus of the heavy chain of a full-length anti-TRAIL-R2 IgG1 monoclonal antibody. Both N- or C-terminal BsAbs were active in inhibiting tumor cell growth in vitro, and with some cell lines demonstrated enhanced activity relative to the combination of parental Abs. Pharmacokinetic studies in mice revealed long serum half-lives for the BsAbs. In murine tumor xenograft models, therapeutic treatment with the BsAbs resulted in reduction in tumor volume either comparable to or greater than the combination of parental antibodies, indicating that simultaneously targeting and cross-linking receptor pairs is an effective strategy for treating tumor cells. These studies support that stability-engineering is an enabling step for producing scalable IgG-like BsAbs with properties desirable for biopharmaceutical development.Key words: bispecific antibodies, single-chain Fv, immunoglobulins, antibody therapeutics, protein stability, pharmacokinetics, protein engineering, tumor inhibition, cancer treatment     

Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties

By simultaneous binding two disease mediators, bispecific antibodies offer the opportunity to broaden the utility of antibody-based therapies. Herein, we describe the design and characterization of Bs4Ab, an innovative and generic bispecific tetravalent antibody platform. The Bs4Ab format comprises a full-length IgG1 monoclonal antibody with a scFv inserted into the hinge domain. The Bs4Ab design demonstrates robust manufacturability as evidenced by MEDI3902, which is currently in clinical development. To further demonstrate the applicability of the Bs4Ab technology, we describe the molecular engineering, biochemical, biophysical, and in vivo characterization of a bispecific tetravalent Bs4Ab that, by simultaneously binding vascular endothelial growth factor and angiopoietin-2, inhibits their function. We also demonstrate that the Bs4Ab platform allows Fc-engineering similar to that achieved with IgG1 antibodies, such as mutations to extend half-life or modulate effector functions.     

A Novel Antibody Engineering Strategy for Making Monovalent Bispecific Heterodimeric IgG Antibodies by Electrostatic Steering Mechanism

Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies.     

Playing the DISC: Turning on TRAIL death receptor-mediated apoptosis in cancer

Formation of the pro-apoptotic death-inducing signaling complex (DISC) can be initiated in cancer cells via binding of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to its two pro-apoptotic receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2. Primary components of the DISC are trimerized TRAIL-R1/-R2, FADD, caspase 8 and caspase 10. The anti-apoptotic protein FLIP can also be recruited to the DISC to replace caspase 8 and form an inactive complex. Caspase 8/10 processing at the DISC triggers the caspase cascade, which eventually leads to apoptotic cell death. Besides TRAIL, TRAIL-R1- or TRAIL-R2-selective variants of TRAIL and agonistic antibodies have been designed. These ligands are of interest as anti-cancer agents since they selectively kill tumor cells. To increase tumor sensitivity to TRAIL death receptor-mediated apoptosis and to overcome drug resistance, TRAIL receptor ligands have already been combined with various therapies in preclinical models. In this review, we discuss factors influencing the initial steps of the TRAIL apoptosis signaling pathway, focusing on mechanisms modulating DISC assembly and caspase activation at the DISC. These insights will direct rational design of drug combinations with TRAIL receptor ligands to maximize DISC signaling.     

Targeting TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), also known as Apo2L, has been investigated in the past decade for its promising anticancer activity due to its ability to selectively induce apoptosis in tumoral cells by binding to TRAIL receptors (TRAIL-R). Macromolecules such as agonistic monoclonal antibodies and recombinant TRAIL have not proven efficacious in clinical studies, therefore several small molecules acting as TRAIL-R agonists are emerging in the scientific literature. In this work we focus on systemizing these drug molecules described in the past years, in order to better understand and predict the requirements for a novel anti-tumoral therapy based on the TRAIL-R-induced apoptotic mechanism.     

Treatment of mice bearing BCL1 lymphoma with bispecific antibodies

Bispecific antibodies with specificity for the CD3/TCR complex of CTL and a target cell Ag can bridge both cell types and trigger cellular cytoxicity. We have produced bispecific antibodies, directed against the surface-expressed Id of the mouse BCL1 lymphoma and the mouse CD3 complex, by hybrid-hybridoma fusion. Two recombination Ig were purified to homogeneity: B1 X 7D6F, which is univalent for Id and CD3 binding and B1 X 7D6M, which is univalent for Id binding but has lost the CD3 binding because of association of the anti-CD3 H chain with the inappropriate L chain. In vitro studies indicate that bridging the TCR/CD3 complex of resting T cells with tumor IgM Id and the appropriate bispecific antibody induced proliferation and secretion of IL-2. Furthermore, in cytotoxicity assays using 51Cr-labeled tumor cells, preactivated T cells could be targeted with the bispecific antibody to give complete lysis of the Ag+ tumor. Finally, the activity of the bispecific antibody was confirmed in vivo. Animals treated i.v. with 5 micrograms of bispecific antibody 9 days after receiving BCL1 cells were cured. Furthermore, when these animals were checked at 150 days for dormant or variant tumors, as have been reported after other forms of immunotherapy in this model, none could be found. Immunotherapy experiments comparing a mixture of control antibodies with the bispecific antibody demonstrate that tumor cell-T cell bridging is established in vivo and is required for therapeutic success. These results indicate the importance of bispecific antibodies as a novel form of treatment for cancer.     

A bispecific antibody effectively neutralizes all four serotypes of dengue virus by simultaneous blocking virus attachment and fusion

Although dengue virus (DENV) infection severely threatens the health of humans, no specific antiviral drugs are currently approved for clinical use against DENV infection. Attachment and fusion are 2 critical steps for the flavivirus infection, and the corresponding functional epitopes are located at E protein domain III (E-DIII) and domain II (E-DII), respectively. Here, we constructed a bispecific antibody (DVD-1A1D-2A10) based on the 2 well-characterized anti-DENV monoclonal antibodies 1A1D-2 (1A1D) and 2A10G6 (2A10). The 1A1D antibody binds E-DIII and can block the virus attaching to the cell surface, while the 2A10 antibody binds E-DII and is able to prevent the virus from fusing with the endosomal membrane. Our data showed that DVD-1A1D-2A10 retained the antigen-binding activity of both parental antibodies. Importantly, it was demonstrated to be significantly more effective at neutralizing DENV than its parental antibodies both in vitro and in vivo, even better than the combination of them. To eliminate the potential antibody-dependent enhancement (ADE) effect, this bispecific antibody was successfully engineered to prevent Fc-γ-R interaction. Overall, we generated a bispecific anti-DENV antibody targeting both attachment and fusion stages, and this bispecific antibody broadly neutralized all 4 serotypes of DENV without risk of ADE, suggesting that it has great potential as a novel antiviral strategy against DENV.