Biparatopic Antibody Research Articles

A biparatopic agonistic antibody that mimics fibroblast growth factor 21 ligand activity

Bispecific antibodies have become important formats for therapeutic discovery. They allow for potential synergy by simultaneously engaging two separate targets and enable new functions that are not possible to achieve by using a combination of two monospecific antibodies. Antagonistic antibodies dominate drug discovery today, but only a limited number of agonistic antibodies (i.e. those that activate receptor signaling) have been described. For receptors formed by two components, engaging both of these components simultaneously may be required for agonistic signaling. As such, bispecific antibodies may be particularly useful in activating multicomponent receptor complexes. Here, we describe a biparatopic (i.e. targeting two different epitopes on the same target) format that can activate the endocrine fibroblast growth factor (FGF) 21 receptor (FGFR) complex containing β-Klotho and FGFR1c. This format was constructed by grafting two different antigen-specific VH domains onto the VH and VL positions of an IgG, yielding a tetravalent binder with two potential geometries, a close and a distant, between the two paratopes. Our results revealed that the biparatopic molecule provides activities that are not observed with each paratope alone. Our approach could help address the challenges with heterogeneity inherent in other bispecific formats and could provide the means to adjust intramolecular distances of the antibody domains to drive optimal activity in a bispecific format. In conclusion, this format is versatile, is easy to construct and produce, and opens a new avenue for agonistic antibody discovery and development.

Abstract LB-A20: Strategies to Improve the Safety and Efficacy of Biparatopic Antibody Drug Conjugates

Abstract Antibody drug conjugates (ADC’s) define an emerging class of targeted therapies for cancer, with over 60 ADC’s currently in clinical development. Although antibodies can exhibit exquisite target specificity, expression of the tumor target in normal tissue can limit the tolerated dose that one can achieve with an ADC. We designed two different affinity variant biparatopic antibodies that recognize distinct epitopes on EGFR and promote receptor clustering that leads to enhanced lysosomal trafficking. These ADC’s comprise two different strategies to target EGFR expressing tumors: a high affinity, potency driven approach and a lowered affinity, threshold driven approach. We show that both ADC’s bind to tumor cells, are rapidly internalized, and traffic to the lysosome, where the warhead is efficiently released by lysosomal hydrolases. In vitro, the high affinity ADC exhibited potent killing of cancer cell lines, with IC50’s in the double digit picomolar range in cells with relative EGFR densities as low as 25,000, while the low affinity ADC was most effective when the EGFR density was greater than 500,000. The high affinity ADC was active in patient derived xenograft (PDX) models derived from colorectal and lung cancer patients harboring clinically relevant oncogenic drivers, such as KRAS and BRAF mutations or EGFR aberrations. This ADC induced tumor stasis or regression, with minimally efficacious doses between 1 and 3 mg/kg, in multiple tumor models across a broad range of EGFR expression (IHC scores ranging from 1 to 3). In a cynomolgus monkey safety study, this ADC showed a manageable safety profile at 2 mg/kg, with skin effects as the dose limiting toxicity. The potency of the high affinity ADC may provide meaningful activity across a range of EGFR expression levels, at doses that are tolerable in the clinic. The lowered affinity ADC exhibited an improved safety profile compared to the high affinity ADC, with a highest non-severely toxic dose (HNSTD) of 6 mg/kg, presumably driven by a reduction in binding to normal tissues that express comparably lower EGFR levels relative to the tumor. This lowered affinity ADC showed a more restricted anti-tumor activity, causing regressions at a minimally efficacious dose between 3 and 5 mg/kg, in PDX models that expressed high levels of EGFR, as judged by IHC scores of 3. The lowered affinity ADC required higher EGFR expression for activity, but the improved tolerability may provide a selectivity threshold that discriminates tumor from normal tissue in patients whose tumors overexpress EGFR. Our findings support the concept that modulating antibody affinity can yield distinct ADC’s that represent different strategies for balancing safety and efficacy. The work presented here provides a conceptual framework for designing unique ADC candidates tailored to the particular expression profile and safety considerations around specific tumor associated targets. Citation Format: Frank I. Comer, Xinzhong Wang, Andy Q. Yuan, Vanessa Muniz-Medina, Agnieszka Sadowska, Lan Xu, Samuel Perry, Pamela Thompson, Ralph Minter, Robert Woods, Mary Jane Hinrichs, Molly Reed, Shameen Affif-Rider, Marlon Rebelatto, Charles Brown, Hui Feng, Rakesh Dixit, Steven Coats, Jane Osbourn. Strategies to Improve the Safety and Efficacy of Biparatopic Antibody Drug Conjugates [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A20.

Abstract 31: Preclinical development of a novel biparatopic HER2 antibody with activity in low to high HER2 expressing cancers

Abstract HER2-directed therapies have improved clinical outcomes for many patients with HER2 overexpressing breast and gastric cancer. Despite these successes, there remains a need to develop effective HER2-targeted therapies for these and other HER2-expressing tumors, particularly in the setting of recurrent or metastatic disease. One approach is to develop a single multifunctional antibody that has improved capacity and efficiency for binding HER2 compared with available HER2 inhibitors, can elicit ADCC, block ligand induced heterodimerization of HER2 with other HER family receptors, and down-regulate HER2 protein on the cell surface. ZW25 is a novel humanized, IgG1-like bispecific antibody directed against two distinct epitopes on the HER2 receptor. ZW25 has low nM binding affinity (0.9-16 nM) to recombinant HER2 and to cultured cancer cells with a range of HER2 expression, and shows higher maximal binding (1.4-1.9x) than monospecific HER2 antibodies on all tumor cell lines tested. The unique structure of ZW25 favors crosslinking of multiple HER2 receptors which is thought to promote clustering, and improves HER2 internalization compared to monospecific HER2 antibodies. In vitro studies demonstrated that ZW25 alone caused significant inhibition in growth of human cancer cell lines with a wide range of HER2 expression (5-54% inhibition depending on the cell line), and inhibited ligand-mediated tumor cell proliferation. ZW25 elicited concentration-dependent ADCC at nM potency (0.05-64 nM) with maximal lysis up to 52% on TNBC cell lines expressing HER2 at a 0/1+ level. ZW25 also exhibited synergy and additivity with multiple chemotherapeutic agents including platins, taxanes, microtubule inhibitors, and DNA synthesis inhibitors in various HER2 expressing tumor cell lines. In vivo studies demonstrated antitumor activity and/or improved survival against xenografts from a range of tumor types. In a HER2 3+ gastric cancer PDX model ZW25 induced significant tumor regressions, exhibiting 307% tumor growth inhibition relative to hIgG control and a 70% complete regression rate (trastuzumab exhibited 111% tumor growth control with no complete regressions). In a HER2 1+ breast cancer PDX model ZW25 significantly improved median survival (58 vs 28 days) and exhibited 141% tumor growth inhibition relative to hIgG control (trastuzumab exhibited 63% tumor growth inhibition, with median survival 39 days). In a low HER2-expressing NSCLC CDX model ZW25 improved median survival compared to hIgG control or cisplatin (> 66 vs 25 or 26 d respectively) and exhibited 159% tumor growth inhibition relative to hIgG control. In a GLP repeat dose toxicology study, ZW25 was well tolerated with no adverse effects at doses up to 150 mg/kg weekly IV for up to 8 weeks. Based on these findings, a first in human clinical trial of ZW25 has been initiated in patients with recurrent and/or metastatic HER2-expressing cancers. Citation Format: Nina Weisser, Grant Wickman, Rupert Davies, Gerry Rowse. Preclinical development of a novel biparatopic HER2 antibody with activity in low to high HER2 expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 31. doi:10.1158/1538-7445.AM2017-31

Targeted Fcγ Receptor (FcγR)-mediated Clearance by a Biparatopic Bispecific Antibody

Soluble ligands have commonly been targeted by antibody therapeutics for cancers and other diseases. Although monoclonal antibodies targeting such ligands can block their interactions with their cognate receptors, they can also significantly increase the half-life of their ligands by FcRn-mediated antibody recycling, thereby evading ligand renal clearance and requiring increasingly high antibody doses to neutralize the increasing pool of target. To overcome this issue, we generated a bispecific/biparatopic antibody (BiSAb) that targets two different epitopes on IL-6 to block IL-6-mediated signaling. The BiSAb formed large immune complexes with IL-6 that can bind Fcγ receptors on phagocytic cells and are rapidly internalized. In addition, rapid clearance of the BiSAb·IL-6 complex was observed in mice while the parental antibodies prolonged the serum half-life of IL-6. Intravital imaging of the liver in mice confirmed that the rapid clearance of these large immune complexes was associated with Fcγ receptor-dependent binding to Kupffer cells in the liver. The approach described here provides a general strategy for therapeutic antibodies with the ability to not only neutralize but also actively drive clearance of their soluble antigens.

Dual anti-idiotypic purification of a novel, native-format biparatopic anti-MET antibody with improved in vitro and in vivo efficacy.

Bispecific antibodies are of great interest due to their ability to simultaneously bind and engage different antigens or epitopes. Nevertheless, it remains a challenge to assemble, produce and/or purify them. Here we present an innovative dual anti-idiotypic purification process, which provides pure bispecific antibodies with native immunoglobulin format. Using this approach, a biparatopic IgG1 antibody targeting two distinct, HGF-competing, non-overlapping epitopes on the extracellular region of the MET receptor, was purified with camelid single-domain antibody fragments that bind specifically to the correct heavy chain/light chain pairings of each arm. The purity and functionality of the anti-MET biparatopic antibody was then confirmed by mass spectrometry and binding experiments, demonstrating its ability to simultaneously target the two epitopes recognized by the parental monoclonal antibodies. The improved MET-inhibitory activity of the biparatopic antibody compared to the parental monoclonal antibodies, was finally corroborated in cell-based assays and more importantly in a tumor xenograft mouse model. In conclusion, this approach is fast and specific, broadly applicable and results in the isolation of a pure, novel and native-format anti-MET biparatopic antibody that shows superior biological activity over the parental monospecific antibodies both in vitro and in vivo.

Abstract 2955: Herceptarg, a novel heterodimeric biparatopic common light chain IgG1 antibody based on trastuzumab and pertuzumab, exerts potent anti-tumoral activity

Abstract Introduction: Trastuzumab and pertuzumab are humanized antibodies recognizing different functional HER2 epitopes. Preclinical data demonstrated strong anti-tumoral efficacy when combining them (Scheuer et al., Canc Res, 2009). The CLEOPATRA trial showed that the addition of pertuzumab to trastuzumab + docetaxel strongly improves overall survival of HER2+ metastatic breast cancer patients (Swain et al., NEJM, 2015). Herceptarg is a novel heterodimeric 1+1 biparatopic common light chain IgG1 antibody based on trastuzumab and pertuzumab. Methods: Using consensus light chains, pertuzumab heavy chain affinity maturation via phage display and knob-into-holes technology, a heterodimeric biparatopic common light chain antibody based on trastuzumab and pertuzumab was generated (Figure 1A). This bispecific antibody was characterized in direct comparison to the respective parental antibodies and their combination in vitro by surface plasmon resonance, proliferation, ADCC and CDC assays and in vivo using the orthotopic KPL-4 breast cancer xenograft model. KPL-4 cells were provided by Prof. Kurebayashi (Kawasaki Medical School, Kurashiki, Japan). Results: In vitro, Herceptarg has the highest binding affinity for HER2 on cells, mediates potent ADCC activity, comparable or superior growth inhibition activity of breast and gastric cancer cells and CDC superior to the combination of pertuzumab and trastuzumab. In vivo, Herceptarg (10 mg/kg, q1w) mediates anti-tumoral efficacy in the orthotopic KPL-4 breast cancer xenograft model resulting in tumor regression comparable to the combination of trastuzumab and pertuzumab (10 mg/kg, q1w, each), and superior to the respective single agent therapies. Conclusions: Taken together, these data demonstrate that Herceptarg, as a single IgG1 bispecific antibody is superior (or at least comparable) to the combination of trastuzumab and pertuzumab in vitro and in vivo and may ultimately improve outcome of breast and gastric cancer patients. Citation Format: Ekkehard Moessner, Thomas Hofer, Inja Waldhauer, Werner Scheuer, Ralf Hosse, Lydia Duerner, Mi He, Karlheinz Zick, Jens Fischer, Claudio Sustmann, Tina Weinzierl, Marina Bacac, Christian Gerdes, Pablo Umana, Christian Klein. Herceptarg, a novel heterodimeric biparatopic common light chain IgG1 antibody based on trastuzumab and pertuzumab, exerts potent anti-tumoral 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 2955.

Abstract 2970: MEDI4276, a HER2-targeting antibody tubulysin conjugate, displays potent in vitro and in vivo activity in preclinical studies

Abstract Antibody drug conjugates (ADCs) combine the specificity of antibodies with the potent cytotoxicity of small molecule drugs and have shown to provide therapeutic options for various cancers. We report herein the discovery of a HER2-targeting ADC MEDI4276 that showed potent cell killing activity in vitro in cancer cell lines that express the HER2 receptor. The observed in vitro activity translated into in vivo tumor growth inhibition in various xenograft mouse models. MEDI4276 is a homogeneous molecule with precise control of drug loading following site specific conjugation of a cytotoxic drug. The drug in MEDI4276 is MMETA, a fully synthetic analog of the tubulysin family that showed pM potency in a panel of cancer cell lines. MMETA was conjugated to the antibody via engineered cysteines with a maleimide-bearing mc-Lys protease cleavable linker. The antibody in MEDI4276 is a bivalent biparatopic antibody targeting two distinct non-overlapping epitopes on HER2 that leads to antibody-receptor clustering following binding and thus promoting internalization, lysosomal trafficking and degradation. The combination of enhanced internalization and potent cytotoxic drug allows for this ADC to kill tumor cell populations with a broader range of HER2 expression. Preclinical studies showed that MEDI4276 induced tumor regression in HER2-positive tumor models that had developed acquired resistance to T-DM1 and in a number of models with lower HER2 expression that are refractory to T-DM1 treatment. Overall, our findings underscore the potential application of MEDI4276 to treat a large patient population that is ineligible for or relapsed/refractory to current HER2-targeted therapies. MEDI4276 is currently being investigated in a Phase I clinical trial. Citation Format: John Li, Dorin Toader, Samuel R. Perry, Vanessa Muniz-Medina, Leslie Wetzel, Marlon C. Rebelatto, Mary Jane Masson Hinrichs, Ryan Fleming, Binyam Bezabeh, Pamela Thompson, Nazzareno Dimasi, Brandon Lam, Xian-Qing Yu, Changshou Gao, Rakesh Dixit, Steven Coats, Jane Osbourn, Herren Wu. MEDI4276, a HER2-targeting antibody tubulysin conjugate, displays potent in vitro and in vivo activity in preclinical studies. [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 2970.

Abstract PD5-08: A biparatopic HER2-targeting antibody-drug conjugate demonstrates potent antitumor activity in primary tumor models that are refractory to or ineligible for HER2-targeted therapies

Abstract Current HER2-targeted drugs are ineffective in killing cancer cells expressing relatively low levels of HER2. Therefore, more than 60% of breast cancer patients are ineligible for HER2-targeted therapies because of lack of HER2 overexpression and the vast majority of eligible patients who initially respond to the treatment will eventually relapse. MedImmune is developing a novel HER2-targeting antibody-drug conjugate (ADC) to address this unmet medical need. We show that a bivalent biparatopic antibody targeting two distinct non-overlapping epitopes on HER2 is able to induce receptor clustering on the tumor cell surface, which in turn facilitates internalization and promotes lysosomal trafficking and degradation. When conjugated with a tubulysin-based microtubule inhibitor, the biparatopic antibody can deliver a greater quantity of cytotoxin into the targeted cancer cells. As a result, it demonstrated superior antitumor activity over Kadcyla® (T-DM1) in HER2-overexpressing (HER2-positive) tumor models. It also induced complete tumor regression in a HER2-positive tumor model that had developed acquired resistance to T-DM1 through chronic exposure. Moreover, to explore the potential clinical applications in treating the HER2 non-overexpressing (HER2-negative) patients the biparatopic ADC was evaluated across 17 primary tumor models derived from HER2-negative breast cancer patients among which 13 were triple-negative. Other criteria were also considered in the selection of these 17 models, including the degree of heterogeneity in HER2 expression, ER/PR status and histopathologic subclass, to maximize the diversity of tumor subtypes in the study. The biparatopic ADC demonstrated potent antitumor activity regardless of the histopathologic subclass and ER/PR status of the tumor. At the dose of 1 mg/kg, 41% of the tumor models (7 out of 17) showed tumor regression and 6% (1 out of 17) showed tumor stasis. At the dose of 3 mg/kg, 71% of the models (12 out of 17) showed tumor regression and 12% (2 out of 17) showed tumor stasis. Overall, our findings underscore the potential use of this novel HER2-targeting ADC to treat a large patient population that is ineligible for or relapsed/refractory to current HER2-targeted therapies, and thus warrant investigation in the clinic. Citation Format: Li JY, Perry SR, Muniz-Medina V, Wetzel LK, Rebelatto MC, Bezabeh BZ, Fleming RL, Dimasi N, Gao C, Wu H, Jenkins DW, Osbourn JK, Coats SR. A biparatopic HER2-targeting antibody-drug conjugate demonstrates potent antitumor activity in primary tumor models that are refractory to or ineligible for HER2-targeted therapies. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD5-08.

A Biparatopic HER2-Targeting Antibody-Drug Conjugate Induces Tumor Regression in Primary Models Refractory to or Ineligible for HER2-Targeted Therapy

Antibody-drug conjugate (ADC) which delivers cytotoxic drugs specifically into targeted cells through internalization and lysosomal trafficking has emerged as an effective cancer therapy. We show that a bivalent biparatopic antibody targeting two non-overlapping epitopes on HER2 can induce HER2 receptor clustering, which in turn promotes robust internalization, lysosomal trafficking, and degradation. When conjugated with a tubulysin-based microtubule inhibitor, the biparatopic ADC demonstrates superior anti-tumor activity over ado-trastuzumab emtansine (T-DM1) in tumor models representing various patient subpopulations, including T-DM1 eligible, T-DM1 ineligible, and T-DM1 relapsed/refractory. Our findings indicate that this biparatopic ADC has promising potential as an effective therapy for metastatic breast cancer and a broader patient population may benefit from this unique HER2-targeting ADC.

EGFR endocytosis requires its kinase activity and N-terminal transmembrane dimerization motif

EGFR signaling is attenuated by endocytosis and degradation of receptor-ligand complexes in lysosomes. Endocytosis of EGFR is known to be regulated by multiple post-translational modifications. The observation that prevention of these modifications does not block endocytosis completely, suggests the involvement of other mechanism(s). Recently, receptor clustering has been suggested to induce internalization of multiple types of membrane receptors. However, the mechanism of clustering-induced internalization remains unknown. We have used biparatopic antibody fragments from llama (VHHs) to induce EGFR clustering without stimulating tyrosine kinase activity. Using this approach, we have found an essential role for the N-terminal GG4-like dimerization motif in the transmembrane domain (TMD) for clustering-induced internalization. Moreover, conventional EGF-induced receptor internalization depends exclusively on this TMD dimerization and kinase activity. Mutations in this dimerization motif eventually lead to reduced EGFR degradation and sustained signaling. We propose a novel role for the TMD dimerization motif in the negative-feedback control of EGFR. The widely conserved nature of GG4-like dimerization motifs in transmembrane proteins suggests a general role for these motifs in clustering-induced internalization.

Anti-EGFR biparatopic-SEED antibody has enhanced combination-activity in a single molecule

Certain combinations of non-competitive anti-EGFR antibodies have been reported to produce new effects on cells compared to either antibody used separately. New and enhanced combination-activity includes increased inhibition of signaling, increased receptor internalization and degradation, reduced proliferation of tumor cell lines and induction of complement-dependent cytotoxicity (CDC) effector function. To test requirements and mechanisms to elicit enhanced combination-activity with different EGFR binding domains, we created an anti-EGFR biparatopic antibody. A biparatopic antibody interacts through two different antigen-binding sites to a single antigen. A heterodimeric antibody with one binding domain derived from the C225 antibody and one binding domain derived from the humanized 425 (hu425) antibody was built on the strand-exchange engineered domain (SEED) scaffold. This anti-EGFR biparatopic-SEED antibody was compared to parental antibodies used alone and in combination, and to the corresponding monovalent anti-EGFR-SEED antibodies used alone or in combination. We found that the anti-EGFR biparatopic-SEED had enhanced activity, similar to the combination of the two parental antibodies. Combinations of monovalent anti-EGFR-SEED antibodies did not produce enhanced effectiveness in cellular assays. Our results show that the anti-EGFR biparatopic antibody created using the SEED scaffold has enhanced combination-activity in a single molecule. Furthermore, these data suggest that the potential to cross-link the two different epitopes is an important requirement in the mechanism of enhanced combination-activity.

Inhibition of tumorigenesis driven by different Wnt proteins requires blockade of distinct ligand-binding regions by LRP6 antibodies

Disregulated Wnt/beta-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer.

Tumor targeting with newly designed biparatopic antibodies directed against two different epitopes of the carcinoembryonic antigen (CEA).

In an attempt to improve tumor targeting and tumor retention time of monoclonal antibodies (MAbs), we prepared biparatopic antibodies (BpAbs) having the capability of binding 2 different non-overlapping epitopes on the same target antigen molecule, namely, the carcinoembryonic antigen (CEA). Six BpAbs were constructed by coupling 2 different Fab' fragments from 4 different specific anti-CEA MAbs recognizing 4 CEA epitopes (Gold 1-4). Demonstration of the double paratopic binding of these antibodies for CEA was confirmed in vitro by inhibition radioimmunoassay and cross-inhibition analysis by surface plasmon resonance (SPR; BIACORE) technology. Using the latter technique, the affinity constants for CEA immobilized onto the sensor chip were found to range from 0.37 to 1.54 x 10(9) M(-1) for the 4 parental F(ab')2 fragments and from 1.88 to 10.14 x 10(9) M(-1) for the BpAbs, demonstrating the advantage of biparatopic binding over conventional F(ab')2 binding. The Ka improvement was particularly high for BpAb F6/35A7 and BpAb F6/B17 with a 9.5- and 8.1-fold increase, respectively, as compared with the parental F(ab')2. In vivo, the 6 BpAbs were compared with their 2 respective parental F(ab')2 by injection of 131I-BpAb/125I-F(ab')2 parental fragments into nude mice xenografted with the human colon carcinoma T380. Dissection 72 hr post-injection demonstrated that BpAb B17/CE25 and BpAb F6/B17 gave higher tumor uptake than that of their parental F(ab')2. This finding is particularly interesting for BpAb F6/B17, which compared favorably with the F6 F(ab')2, one of the best parental F(ab')2 fragments used in our study.

Tumor targeting with newly designed biparatopic antibodies directed against two different epitopes of the carcinoembryonic antigen (CEA)

Tumor targeting with newly designed biparatopic antibodies directed against two different epitopes of the carcinoembryonic antigen (CEA)