Antibody Candidates Research Articles

Ultrasensitive NIR-SERRS Probes with Multiplexed Ratiometric Quantification for In Vivo Antibody Leads Validation.

Immunotargeting ability of antibodies may show significant difference between in vitro and in vivo. To select antibody leads with high affinity and specificity, it is necessary to perform in vivo validation of antibody candidates following in vitro antibody screening. Herein, a robust in vivo validation of anti-tetraspanin-8 antibody candidates against human colon cancer using ratiometric quantification method is reported. The validation is performed on a single mouse and analyzed by multiplexed surface-enhanced Raman scattering using ultrasensitive and near infrared (NIR)-active surface-enhanced resonance Raman scattering nanoprobes (NIR-SERRS dots). The NIR-SERRS dots are composed of NIR-active labels and Au/Ag hollow-shell assembled silica nanospheres. A 93% of NIR-SERRS dots is detectable at a single-particle level and signal intensity is 100-fold stronger than that from nonresonant molecule-labeled spherical Au NPs (80 nm). The result of SERRS-based antibody validation is comparable to that of the conventional method using single-photon-emission computed tomography. The NIR-SERRS-based strategy is an alternate validation method which provides cost-effective and accurate multiplexing measurements for antibody-based drug development.

Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy

ABSTRACT Both in vivo data in preclinical cancer models and in vitro data with T cells from patients with advanced cancer support a role for Tim-3 blockade in promoting effective anti-tumor immunity. Consequently, there is considerable interest in the clinical development of antibody-based therapeutics that target Tim-3 for cancer immunotherapy. A challenge to this clinical development is the fact that several ligands for Tim-3 have been identified: galectin-9, phosphatidylserine, HMGB1, and most recently, CEACAM1. These observations raise the important question of which of these multiple receptor:ligand relationships must be blocked by an anti-Tim-3 antibody in order to achieve therapeutic efficacy. Here, we have examined the properties of anti-murine and anti-human Tim-3 antibodies that have shown functional efficacy and find that all antibodies bind to Tim-3 in a manner that interferes with Tim-3 binding to both phosphatidylserine and CEACAM1. Our data have implications for the understanding of Tim-3 biology and for the screening of anti-Tim-3 antibody candidates that will have functional properties in vivo.

Antibody Selection for Cancer Target Validation of FSH-Receptor in Immunohistochemical Settings.

Background: The follicle-stimulating hormone (FSH)-receptor (FSHR) has been reported to be an attractive target for antibody therapy in human cancer. However, divergent immunohistochemical (IHC) findings have been reported for FSHR expression in tumor tissues, which could be due to the specificity of the antibodies used. Methods: Three frequently used antibodies (sc-7798, sc-13935, and FSHR323) were validated for their suitability in an immunohistochemical study for FSHR expression in different tissues. As quality control, two potential therapeutic anti-hFSHR Ylanthia® antibodies (Y010913, Y010916) were used. The specificity criteria for selection of antibodies were binding to native hFSHR of different sources, and no binding to non-related proteins. The ability of antibodies to stain the paraffin-embedded Flp-In Chinese hamster ovary (CHO)/FSHR cells was tested after application of different epitope retrieval methods. Results: From the five tested anti-hFSHR antibodies, only Y010913, Y010916, and FSHR323 showed specific binding to native, cell-presented hFSHR. Since Ylanthia® antibodies were selected to specifically recognize native FSHR, as required for a potential therapeutic antibody candidate, FSHR323 was the only antibody to detect the receptor in IHC/histochemical settings on transfected cells, and at markedly lower, physiological concentrations (ex., in Sertoli cells of human testes). The pattern of FSH323 staining noticed for ovarian, prostatic, and renal adenocarcinomas indicated that FSHR was expressed mainly in the peripheral tumor blood vessels. Conclusion: Of all published IHC antibodies tested, only antibody FSHR323 proved suitable for target validation of hFSHR in an IHC setting for cancer. Our studies could not confirm the previously reported FSHR overexpression in ovarian and prostate cancer cells. Instead, specific overexpression in peripheral tumor blood vessels could be confirmed after thorough validation of the antibodies used.

Determination of cross-reactivity of poly- and monoclonal antibodies for synthetic cannabinoids by direct SPR and ELISA

One of the main reasons for the rise in popularity of synthetic cannabinoids (SCs) is their ability to remain unrecognized in conventional drug screenings. Due to their structural diversity, caused by the constant introduction of new substances to circumvent legal regulation, antibodies with a wide range of cross-reactivity are necessary for the establishment of a reliable immunological based drug test. Therefore, high-quality binding data are needed to select promising antibody candidates for further development.In this study, we carried out a direct surface plasmon resonance (SPR) method and evaluated its suitability for the characterization of antibody–SC interactions. The cross-reactivity of 22 SCs with three polyclonal antibodies, raised against JWH-018 haptens with different attachment positions of the linker, and two commercial available monoclonal antibodies were determined. These results were compared with the commonly used competitive enzyme-linked immunosorbent assay (ELISA). It could be demonstrated, that direct SPR and competitive ELISA show comparable specificity results for the majority of the measured compounds. However, the reduced manual labor, the real-time analysis and the high information content about the binding events of SPR compared to ELISA, showed that SPR is a valuable tool during the development of antibodies against synthetic cannabinoids, currently the largest group of new psychoactive substances.

Rapid and accurate in silico solubility screening of a monoclonal antibody library

Antibodies represent essential tools in research and diagnostics and are rapidly growing in importance as therapeutics. Commonly used methods to obtain novel antibodies typically yield several candidates capable of engaging a given target. The development steps that follow, however, are usually performed with only one or few candidates since they can be resource demanding, thereby increasing the risk of failure of the overall antibody discovery program. In particular, insufficient solubility, which may lead to aggregation under typical storage conditions, often hinders the ability of a candidate antibody to be developed and manufactured. Here we show that the selection of soluble lead antibodies from an initial library screening can be greatly facilitated by a fast computational prediction of solubility that requires only the amino acid sequence as input. We quantitatively validate this approach on a panel of nine distinct monoclonal antibodies targeting nerve growth factor (NGF), for which we compare the predicted and measured solubilities finding a very close match, and we further benchmark our predictions with published experimental data on aggregation hotspots and solubility of mutational variants of one of these antibodies.

High-throughput screening of hybridoma supernatants using multiplexed fluorescent cell barcoding on live cells

With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development.We have developed a novel technique called Multiplexed Fluorescent Cell Barcoding (MFCB), a flow cytometry-based method based upon the Fluorescent Cell Barcoding (FCB) technique and the Luminex fluorescent bead array system, but is applicable to high-through mAb screens on live cells. Using this technique in our system, we can simultaneously identify or characterize the antibody-antigen binding of up to nine unique fluorescent labeled cell populations in the time that it would normally take to process a single population. This has significantly reduced the amount of time needed for the identification of potential lead candidates.This new technology enables investigators to conduct large-scale primary hybridoma screens using flow cytometry. This in turn has allowed us to screen antibodies more efficiently than before and streamline identification and characterization of lead molecules.

High-Throughput, Multispecies, Parallelized Plasma Stability Assay for the Determination and Characterization of Antibody-Drug Conjugate Aggregation and Drug Release.

The stability of antibody–drug conjugates (ADCs) in circulation is critical for maximum efficacy and minimal toxicity. An ADC reaching the intended target intact can deliver the highest possible drug load to the tumor and reduce off-target toxicity from free drug in the blood. As such, assessment of ADC stability is a vital piece of data during development. However, traditional ADC stability assays can be manually intensive, low-throughput, and require large quantities of ADC material. Here, we introduce an automated, high-throughput plasma stability assay for screening drug release and aggregation over 144 h for up to 40 ADCs across five matrices simultaneously. The amount of ADC material during early drug development is often limited, so this assay was implemented in 384-well format to minimize material requirements to <100 μg of each ADC and 100 μL of plasma per species type. Drug release and aggregation output were modeled using nonlinear regression equations to calculate formation rates for each data type. A set of 15 ADCs with different antibodies and identical valine–citrulline–p-aminobenzylcarbamate–monomethylauristatin E linker-drug payloads was tested and formation rates were compared across ADCs and between species, revealing several noteworthy trends. In particular, a wide range in aggregation was found when altering only the antibody, suggesting a key role for plasma stability screening early in the development process to find and remove antibody candidates with the potential to create unstable ADCs. The assay presented here can be leveraged to provide stability data on new chemistry and antibody screening initiatives, select the best candidate for in vivo studies, and provide results that highlight stability issues inherent to particular ADC designs throughout all stages of ADC development.

Evaluation of two lead malaria transmission blocking vaccine candidate antibodies in natural parasite-vector combinations

Transmission blocking vaccines (TBV) which aim to control malaria by inhibiting human-to-mosquito transmission show considerable promise though their utility against naturally circulating parasites remains unknown. The efficacy of two lead candidates targeting Pfs25 and Pfs230 antigens to prevent onwards transmission of naturally occurring parasites to a local mosquito strain is assessed using direct membrane feeding assays and murine antibodies in Burkina Faso. The transmission blocking activity of both candidates depends on the level of parasite exposure (as assessed by the mean number of oocysts in control mosquitoes) and antibody titers. A mathematical framework is devised to allow the efficacy of different candidates to be directly compared and determine the minimal antibody titers required to halt transmission in different settings. The increased efficacy with diminishing parasite exposure indicates that the efficacy of vaccines targeting either Pfs25 or Pfs230 may increase as malaria transmission declines. This has important implications for late-stage candidate selection and assessing how they can support the drive for malaria elimination.

Abstract 2661: A broad approach for the selection of therapeutic mAbs to TNFR-superfamily members for use in combination with an anti-PD-1 mAb

Abstract The rise of therapeutic antibodies directed towards T cell checkpoint inhibitors has paved the way to a novel and exciting frontier of cancer treatments. Spearheaded by anti-CTLA-4 and PD-1/PD-L1 inhibitors, many investigators have strived to uncover combinatory therapies that could synergize with CTLA-4 and PD-1 inhibitors to improve the overall survival of cancer patients. The TNFR-superfamily has been seen as an ideal combinatory target in providing co-stimulatory T cell signals in sync with the unleashing of T cells from their CTLA-4 and PD-1 brakes. The ideal outcome of these combinations is the induction of T cell memory and effector function towards tumour cells. Current clinical-stage agents against TNFR-superfamily targets include conventional or Fc-engineered therapeutic antibodies and multimeric ligands that aim to cluster and activate targets such as OX40, 4-1BB, GITR, and CD27. For OX40 and GITR, wildtype IgG1 variants offer target cell depletion of OX40/GITR-high expressing Treg cells, and also provide agonism through FcgR-mediated crosslinking. We have taken a broad approach of screening IgG1, IgG2, Fc-engineered and tetravalent antibodies to select for potent therapeutic antibody candidates to TNFR-superfamily targets, and conducted humanized mouse studies as a screen for candidate potency. Citation Format: Cheng Chen, Xiaoniu Miao, Yao Yan, Liang Tang, Bingliang Chen, Junjian Liu, Xiaolin Liu, Michael Yu, Andy Tsun. A broad approach for the selection of therapeutic mAbs to TNFR-superfamily members for use in combination with an anti-PD-1 mAb [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 2661. doi:10.1158/1538-7445.AM2017-2661

Abstract 2136: Inhibiting cancer growth by targeting the TNFR2 oncogene with TNFR2 antagonistic antibodies

Abstract Background: Although antibody targeting of the HER2 oncogene represents an outstanding clinical treatment and a success story showing direct cancer killing with antibodies, many oncogenes are intracellular and are not expressed broadly, whether on a single or multiple tumor types. The recently discovered TNFR2 oncogene is broadly expressed on many human tumors. Colon cancer cells, multiple myeloma cells, renal cell carcinoma cells, Hodgkin’s lymphoma cells, ovarian cancer epithelial cells and cutaneous non-Hodgkin’s lymphoma cells can aberrantly express the TNFR2 receptor as an oncogene for growth. For non-cutaneous T cell lymphomas, the genetic basis of the TNFR2 deregulation has recently been tied to constitutive overexpression of TNFR2 from frequent gene duplications or cytoplasmic TNFR2 mutations that confer constitutive agonism, i.e. tumor expansion. These features make TNFR2 an advantageous molecular target for direct tumor targeting. Methods: We designed monoclonal antibodies to target the TNFR2 oncogene and directly kill human tumor cells. TNFR2-directed antibodies were screened for their ability to induce the death of rapidly growing tumor cells, such as ovarian cancer cells (i.e., OVCAR3). Results: Novel dominant anti-TNFR2 antibody candidates (TNFR2 antagonistic antibodies) did not require Fc binding for activity, expressed dominance over TNF-mediated agonism, and hampered intracellular NF-κB activation and phosphorylation obligatory for TNFR2 signaling and cell growth of tumor cells. Even low doses of TNFR2 antagonists rapidly and directly killed TNFR2 oncogene-expressing ovarian cancer cells. Examination of the structural biology of these dominant TNFR2 antagonist antibodies uncovered a unique and stabilizing TNFR2 receptor formation, anti-parallel dimeric TNFR2, which inhibits intracellular signaling, cannot bind TNF, cannot be cleaved to create soluble TNFR2 and is exponentially more active on the dividing cells of cancer. Conclusions: TNFR2 is a unique and broadly expressed human oncogene that can potentially be targeted to directly stop the growth of cancer cells (including ovarian cancer cells) by antibody-induced cell death. The creation of dominant TNFR2 antagonism provides a unique, non-signaling complex that has implications for the therapeutic targeting of TNF superfamily receptors, especially TNFR2. Citation Format: Heather Torrey, John Butterworth, Toshiyuki Mera, Yoshiaki Okubo, Limei Wang, Danielle Baum, Audrey Defusco, Sara Plager, Sarah Warden, Daniel Huang, Eva Vanamee, Rosemary Foster, Denise L. Faustman. Inhibiting cancer growth by targeting the TNFR2 oncogene with TNFR2 antagonistic antibodies [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 2136. doi:10.1158/1538-7445.AM2017-2136

Abstract 3640: Novel humanized anti-Sialyl-Tn, anti-CD3 bispecific antibodies demonstrate tumor and T-cell specificity for immune activation at the tumor site

Abstract Tumor-associated carbohydrate antigens (TACAs) historically have been challenging targets for antibody therapeutics. Sialyl-Tn (STn) is a cancer specific antigen that is expressed on the surface of carcinomas including ovarian, colon, prostate, and pancreatic tumors but is rarely present in normal tissue. STn expression has been linked to innate immune suppression, a chemoresistant phenotype, metastasis, and poor prognosis. Previous attempts to target this antigen in the clinic with synthetic glycan vaccines proved safe but lacked efficacy. We have developed humanized bispecific antibodies targeting STn and CD3 for T-cell recruitment and activation at the tumor site. These bispecific antibodies were selected for optimal tumor targeting using our glycan microarray that enriches for candidates whose binding is protein-independent and glycan specific. STn-selective binding was demonstrated. Current lead candidates exhibited low nanomolar EC50 binding in flow cytometric assays against both STn expressing tumor cells and T cells. Quantification of T-cell activation and T-cell induced tumor killing in vitro provides a basis for the further clinical development of these bispecific antibody candidates. Citation Format: David A. Eavarone, Jillian Prendergast, Patricia E. Rao, Jenna Stein, Jeff Behrens, Daniel T. Dransfield. Novel humanized anti-Sialyl-Tn, anti-CD3 bispecific antibodies demonstrate tumor and T-cell specificity for immune activation at the tumor site [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 3640. doi:10.1158/1538-7445.AM2017-3640

Development of a Rapid Identification Method for a Variety of Antibody Candidates Using High-throughput Sequencing

As an alternative to hybridoma technology, the antibody phage library system can also be used for antibody selection. This method enables the isolation of antigen-specific binders through an in vitro selection process known as biopanning. While it has several advantages, such as an avoidance of animal immunization, the phage cloning and screening steps of biopanning are time-consuming and problematic. Here, we introduce a novel biopanning method combined with high-throughput sequencing (HTS) using a next-generation sequencer (NGS) to save time and effort in antibody selection, and to increase the diversity of acquired antibody sequences. Biopannings against a target antigen were performed using a human single chain Fv (scFv) antibody phage library. VH genes in pooled phages at each round of biopanning were analyzed by HTS on a NGS. The obtained data were trimmed, merged, and translated into amino acid sequences. The frequencies (%) of the respective VH sequences at each biopanning step were calculated, and the amplification factor (change of frequency through biopanning) was obtained to estimate the potential for antigen binding. A phylogenetic tree was drawn using the top 50 VH sequences with high amplification factors. Representative VH sequences forming the cluster were then picked up and used to reconstruct scFv genes harboring these VHs. Their derived scFv-Fc fusion proteins showed clear antigen binding activity. These results indicate that a combination of biopanning and HTS enables the rapid and comprehensive identification of specific binders from antibody phage libraries.

Abstract 1645: CEACAM1-blockade for T-cell activation and antitumor T-cell response

Abstract Immune checkpoint blockade became the most powerful way to control cancer without causing tremendous side effects, since anti-PD-1 blocking antibodies proved their superiority in clinical outcomes to those of traditional chemo- or targeted therapeutics. CEACAM1 (CCM1) is one of the several immune checkpoint receptors expressed on T cells that mediate suppression of inflammatory T cell response. CCM1 caught our attention for a cancer immune-therapeutic target, because of its inhibitory role in TCR proximal signaling complex. The anti-cancer therapeutic potential of CCM1-blockade has already been studied in mouse models, and one of the anti-human CCM1 antibody clones recently emerged in clinical Phase I studies. CCM1-targeting antibody candidates were screened out of our in-house synthetic libraries, among which a few of the clones stood out showing their significant blocking activities against CCM1-CCM1 homophilic interaction, enhancement of T cell activation, cytokine release, NFAT signaling, and tumor lysis. One of the clones (C25) showed its strongest efficacy equivalent to that of a reference Ab in CCM1-dependent T cell activation, proliferation, and IL-2 release, in parental Jurkat cells as well as in CCM1-overexpressing stable Jurkat cells. Moreover, C25 also enhanced tumor lysis by CCM1+ NK cells or CCM1+ cytotoxic T cells in a CCM1-dependent manner. In addition, C25 shows distinct characteristics in binding to CCM family from those of a reference Ab. Thus, C25 is thought to have a strong therapeutic potential on CCM1+ cancer. Citation Format: So-Young Eun. CEACAM1-blockade for T-cell activation and antitumor T-cell response [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 1645. doi:10.1158/1538-7445.AM2017-1645

Abstract 3330: MUC1* targeting CAR T

Abstract Purpose: Over 75% of all solid tumor cancers are characterized by aberrant expression of MUC1. Yet to date, no effective MUC1 targeting therapeutic has succeeded. The purpose of this body of work was to develop an antibody therapeutic that would effectively treat MUC1 positive solid tumor cancers, without damaging healthy MUC1 positive tissues. Experimental Procedures: MUC1 is a heavily glycosylated transmembrane protein whose expression is restricted to the apical border of healthy epithelial tissues but uniformly expressed over cancerous tissues. We discovered that the cancer-associated form of MUC1 is transmembrane cleavage product that remains after cleavage and release of the bulk of the extracellular domain. We named cleaved MUC1, MUC1* (muk 1 star), and showed that it is a growth factor receptor that is activated by ligand-induced dimerization of its small extracellular domain. We also showed that 100% of pluripotent human stem cells express MUC1* but cleavage stops with the onset of differentiation. By studying stem cells and cancer cells in parallel, we sought to develop antibodies that would only recognize MUC1* as it is expressed on cancerous tissues but would not recognize MUC1* as it exists on some healthy cells and tissues. Summary New Data: We previously showed that we had developed antibodies that recognized the cleaved, growth factor receptor form, MUC1* but not full length MUC1. However, MUC1 is cleaved to a healthy form of MUC1* on, for example, pluripotent stem cells, hematopoietic stem cells and stem cells of intestinal crypts. We now report that we have developed monoclonal antibodies that only recognize the cancerous form of MUC1* and do not recognize these healthy forms of MUC1*. To demonstrate that we have succeeded in deciphering the differences between cancerous MUC1* and healthy MUC1*, we also developed a set of monoclonal antibodies that only recognize healthy MUC1*. Thousands of human cancerous versus normal tissue specimens attest to the specificity of these antibodies. Cancer-specific anti-MUC1* antibodies (Fabs) alone blocked the growth of MUC1* positive breast and hormone refractory prostate cancers in animals, with no detectable adverse effects. On the basis of tissue safety studies and efficacy studies, a clinical candidate antibody MNC2 has been identified, humanized and incorporated into more than 20 CAR constructs. huMNC2-scFv-CAR T cells display typical cytokine release only when co-cultured with MUC1* positive cancer cells. huMNC2-scFv-CAR T cells selectively kill MUC1* positive cancers, while stimulating T cell expansion. Conclusions: The relevant target for anti-cancer drugs is the extracellular domain of MUC1*, not full length MUC1. MUC1* is a growth factor receptor that is activated by NME family growth factors. Subtle differences between MUC1* as expressed on healthy stem-like cells and MUC1* expressed on cancerous cells has allowed the development of cancer-specific MUC1* antibodies, especially suited for cancer immunotherapies. Citation Format: Cynthia Carol Bamdad, Andrew K. Stewart, Benoit J. Smagghe, Luke T. Deary, Victoria L. Kohler, Jared L. Dietz. MUC1* targeting CAR T [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 3330. doi:10.1158/1538-7445.AM2017-3330

Abstract 578: Discovery and characterization of novel antagonistic antibodies that bind with high affinity to human, cynomolgus, and murine TIGIT, an immune checkpoint receptor

Abstract TIGIT is a recently identified coinhibitory immune checkpoint receptor expressed on NK, effector T, and regulatory T cells. In the oncology setting, TIGIT is upregulated on tumor infiltrating immune cells and is co-expressed with exhaustion markers including PD-1, TIM-3 and LAG-3 on infiltrates. TIGIT binds at least two ligands, CD155 and CD112, which are expressed on antigen presenting cells and other tissues, including tumor cells. These ligands also bind the activating receptor CD226, often co-expressed with TIGIT, creating a network that modulates adaptive and innate immune response in a manner analogous to the CD28-CTLA4-CD80-CD86 network. The absence of TIGIT signaling, resulting from genetic deficiency or blockade, enhances anti-tumor immunity in murine models, suggesting that disruption of TIGIT signaling may have clinical utility. To explore this concept, yeast antibody display was used to identify fully human, anti-TIGIT antibodies that block binding to ligands. Multiple rounds of selection with human and mouse TIGIT protein were performed to promote species cross-reactivity, diversity and affinity. A pool of 695 unique clones were screened for binding to TIGIT protein; 65 clones were then selected for further evaluation. Of the 65, 63 competed with CD155 for binding to TIGIT in a ForteBio screen. Fifty-three clones bound cyno TIGIT and 25 bound TIGIT from all three species (human, cyno, mouse). Antibodies bound endogenous TIGIT on primary T cells and blocked binding of ligands to cell surface expressed TIGIT in a dose dependent manner. Twelve clones showed functional activity in a TIGIT blockade bioassay and showed synergy with anti-PD-1 antibody in a PD-1/TIGIT combination bioassay. Activity in the bioassays correlated with affinity for recombinant and cell surface expressed TIGIT. Based on species cross-reactivity, binding affinity and activity in the bioassays, a lead candidate antibody was selected and produced as mouse IgG1 and IgG2a chimeras for testing in mouse tumor models. The chimeric antibodies behaved similarly to the parent clone in vitro exhibiting high affinity for TIGIT, competition with ligand for binding to TIGIT, and functional blockade of CD155-TIGIT interaction. Evaluation of the chimeric anti-TIGIT candidates alone and in combination with anti-PD-1 antibody in mouse syngeneic tumor models is ongoing, and results will be reported at the meeting. Antibody mediated blockade of coinhibitory immunoreceptors has proven clinically efficacious and supports the development of antibodies that target TIGIT. The unique human, non-human primate, and murine cross-reactive TIGIT-specific antibodies described here offer a simplified preclinical development path and the functional activity of these molecules supports their consideration as candidates for therapeutic development. Citation Format: Julia C. Piasecki, Kenneth Brasel, Robert Rosler, Kevin M. Klucher, Scott R. Peterson. Discovery and characterization of novel antagonistic antibodies that bind with high affinity to human, cynomolgus, and murine TIGIT, an immune checkpoint receptor [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 578. doi:10.1158/1538-7445.AM2017-578

A Formulation Development Approach to Identify and Select Stable Ultra–High-Concentration Monoclonal Antibody Formulations With Reduced Viscosities

High protein concentration formulations are required for low-volume administration of therapeutic antibodies targeted for subcutaneous, self-administration by patients. Ultra-high concentrations (≥150 mg/mL) can lead to dramatically increased solution viscosities, which in turn can lead to stability, manufacturing, and delivery challenges. In this study, various categories and individual types of pharmaceutical excipients and other additives (56 in total) were screened for their viscosity reducing effects on 2 different mAbs. The physicochemical stability profile, as well as viscosity ranges, of several candidate antibody formulations, identified and designed based on the results of the excipient screening, were evaluated over a 6-month time period under accelerated and real-time storage conditions. In addition to reducing the solution viscosities to acceptable levels for parenteral administration (using currently available and acceptable delivery devices), the candidate formulations did not result in notable losses of physicochemical stability of the 2 antibodies on storage for 6 months at 25°C. The experiments described here demonstrate the feasibility of a formulation development and selection approach to identify candidate high-concentration antibody formulations with viscosities within pharmaceutically acceptable ranges that do not adversely affect their physicochemical storage stability.

Highly Specific and Effective Targeting of EGFRvIII-Positive Tumors with TandAb Antibodies.

To harness the cytotoxic capacity of immune cells for the treatment of solid tumors, we developed tetravalent, bispecific tandem diabody (TandAb) antibodies that recognize EGFRvIII, the deletion variant III of the epidermal growth factor receptor (EGFR), and CD3 on T-cells, thereby directing immune cells to eliminate EGFRvIII-positive tumor cells. Using phage display, we identified scFv antibodies selectively binding to EGFRvIII. These highly EGFRvIII-specific, fully human scFv were substantially improved by affinity maturation, achieving KDs in the picomolar range, and were used to construct a set of bispecific EGFRvIII-targeting TandAbs with a broad range of binding and cytotoxic properties. These antibodies exhibited an exquisite specificity for a distinguished epitope in the N-terminal portion of EGFRvIII, as shown on recombinant antigen in Western Blot, SPR, and ELISA, as well as on antigen-expressing cells in FACS assays, and did not bind to the wild-type EGFR. High-affinity EGFRvIII/CD3 TandAbs were most potent in killing assays, displaying cytotoxicity toward EGFRvIII-expressing CHO, F98 glioma, or human DK-MG cells with EC50 values in the range of 1–10 pM in vitro. They also demonstrated dose-dependent growth control in vivo in an EGFRvIII-positive subcutaneous xenograft tumor model. Together with the tumor-exclusive expression of EGFRvIII, the EGFRvIII/CD3 TandAbs’ high specificity and strictly target-dependent activation with no off-target activity provide an opportunity to target tumor cells and spare normal tissues, thereby reducing the side effects associated with other anti-EGFR therapies. In summary, EGFRvIII/CD3 TandAbs are highly attractive therapeutic antibody candidates for selective immunotherapy of EGFRvIII-positive tumors.

Comparative study of therapeutic antibody candidates derived from mini-pool and clonal cell lines.

The long journey of developing a drug from initial discovery target identification to regulatory approval often leaves many patients with missed window of opportunities. Both regulatory agencies and biopharmaceutical industry continue to develop creative approaches to shorten the time of new drug development in order to deliver life-saving medicine to patients. Generally, drug substance materials to support the toxicology and early phase clinical study can only be manufactured after creating the final Master Cell Bank (MCB) of the clonally derived cell line, which normally takes 1-2 years. With recent advances in cell line development, cell culture process and analytical technologies, generating more homogeneous bulk/mini-pool population with higher productivity and acceptable quality attributes has become a norm, thereby making it possible to shorten the timeline to initiate First in Human (FIH) trial by using bulk/mini-pool generated materials to support toxicology and FIH studies. In this study, two monoclonal antibodies of different subclasses (IgG1 and IgG4) were expressed from the mini-pool cells as well as clonally derived cell lines generated from the same mini-pool. Cell growth, productivity, and product quality were compared between the materials generated from the mini-pool and clonally derived cell line. The results demonstrate the similarity of the antibody products generated from mini-pool cells and clonally derived cell lines from the same mini-pool, and strongly support the concept and feasibility of using antibody materials produced from mini-pool cultures for toxicology and FIH studies. The strategy to potentially shorten the FIH timeline is discussed. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1456-1462, 2017.

Development of a CHO-Based Cell-Free Platform for Synthesis of Active Monoclonal Antibodies.

Chinese Hamster Ovary (CHO) cells are routinely optimized to stably express monoclonal antibodies (mAbs) at high titers. At the early stages of lead isolation and optimization, hundreds of sequences for the target protein of interest are screened. Typically, cell-based transient expression technology platforms are used for expression screening, but these can be time- and resource-intensive. Here, we have developed a cell-free protein synthesis (CFPS) platform utilizing a commercially available CHO extract for the rapid in vitro synthesis of active, aglycosylated mAbs. Specifically, we optimized reaction conditions to maximize protein yields, established an oxidizing environment to enable disulfide bond formation, and demonstrated the importance of temporal addition of heavy chain and light chain plasmids for intact mAb production. Using our optimized platform, we demonstrate for the first time to our knowledge the cell-free synthesis of biologically active, intact mAb at >100 mg/L using a eukaryotic-based extract. We then explored the utility of our system as a tool for ranking yields of candidate antibodies. Unlike stable or transient transfection-based screening, which requires a minimum of 7 days for setup and execution, results using our CHO-based CFPS platform are attained within 2 days and it is well-suited for automation. Further development would provide a tool for rapid, high-throughput prediction of mAb expression ranking to accelerate design-build-test cycles required for antibody expression and engineering. Looking forward, the CHO-based CFPS platform could facilitate the synthesis of toxic proteins as well.

Impact of antibody subclass and disulfide isoform differences on the biological activity of CD200R and βklotho agonist antibodies

Agonism of cell surface receptors by monoclonal antibodies is dependent not only on its ability to bind the target, but also to deliver a biological signal through receptors to the cell. Immunoglobulin G2 antibodies (IgG2s) are made up of a mixture of distinct isoforms (IgG2-A, -B and A/B), which differ by the disulfide connectivity at the hinge region. When evaluating panels of agonistic antibodies against CD200 receptor (CD200R) or βklotho receptor (βklotho), we noticed striking activity differences of IgG1 or IgG2 antibodies with the same variable domains. For the CD200R antibody, the IgG2 antibody demonstrated higher activity than the IgG1 or IgG4 antibody. More significantly, for βklotho, agonist antibodies with higher biological activity as either IgG2 or IgG1 were identified. In both cases, ion exchange chromatography was able to isolate the bioactivity to the IgG2-B isoform from the IgG2 parental mixture. The subclass-related increase in agonist activity was not correlated with antibody aggregation or binding affinity, but was driven by enhanced avidity for the CD200R antibody. These results add to the growing body of evidence that show that conformational differences in the antibody hinge region can have a dramatic impact on the antibody activity and must be considered when screening and engineering therapeutic antibody candidates. The results also demonstrate that the IgG1 (IgG2-A like) or the IgG2-B form may provide the most active form of agonist antibodies for different antibodies and targets.