A CD25×TIGIT bispecific antibody induces anti-tumor activity through selective intratumoral Treg cell depletion.

Intratumoral regulatory T cells (Tregs) express high levels of CD25 and TIGIT, which are also recognized as markers of effector T cell (Teff) activation. Targeting these molecules each alone with monoclonal antibodies (mAbs) poses a risk of concurrently depleting both Teffs and peripheral Tregs, thereby compromising the effectiveness and selectivity of intratumoral Treg depletion. Here, leveraging the increased abundance of CD25+ TIGIT+ double-positive Tregs in the solid tumor microenvironment (but not in peripheral tissues), we explore the feasibility of using a CD25×TIGIT bispecific antibody (bsAb) to selectively deplete intratumoral Tregs. We initially constructed a bsAb co-targeting mouse CD25 and TIGIT, NSWm7210, and found that NSWm7210 conferred enhanced intratumoral Treg depletion, Teff activation, and tumor suppression as compared to the parental monotherapies in mouse models. We subsequently constructed a bsAb co-targeting human CD25 and TIGIT (NSWh7216), which preferentially eliminated CD25+ TIGIT+ double-positive cells over single-positive cells in vitro. NSWh7216 exhibited enhanced anti-tumor activity without toxicity of peripheral Tregs in CD25 humanized mice compared to the parental monotherapies. Our study illustrates the use of CD25×TIGIT bsAbs as effective agents against solid tumors based on selective depletion of intratumoral Tregs.

Monitoring of the disulfide scrambled species by mixed-mode SEC-HPLC during the purification of a bispecific antibody.

Size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) is an analytical method routinely used for assessing aggregation content in protein samples. As SEC-HPLC separates analytes based on their hydrodynamic radius, it generally lacks the capability of differentiating species that are similar in size. Recently while purifying a bispecific antibody (bsAb), we noticed that SEC-HPLC can provide certain degree of resolution between the target bsAb and a disulfide scrambled form, although these two species were identical in molecular weight. In seeing the unexpected potential of SEC-HPLC at resolving species with similar size, we further tested Zenix SEC-300, a mixed-mode SEC-HPLC column from Sepax, which was reported to be capable of separating protein analytes based on other factors besides size. The Zenix column indeed provided resolution much better than the regular SEC-HPLC column. Upon further optimization, the Zenix column allowed close to baseline separation of the correctly folded and the disulfide scrambled species. The current study, as a complement to the previous reports, further demonstrates that mix-mode SEC-HPLC is capable of separating protein analytes that are close in size but are different in conformation and/or surface characteristics.

CaptureSelect FcXP affinity medium exhibits strong aggregate separation capability.

Protein A affinity chromatography has been widely used for initial product capture in recombinant antibody/Fc-fusion purification. However, in general Protein A lacks the capability of separating aggregates (unless the aggregates are too large to enter the pores of resin beads or have their Protein A binding sites buried, in which case the aggregates do not bind). In the current work, we demonstrated that CaptureSelect FcXP affinity medium exhibited strong aggregate separation capability and effectively removed aggregates under pH or conductivity gradient elution in two bispecific antibody (bsAb) cases. For these two cases, aggregate contents were reduced from >16% and >22% (in the feed) to <1% and <5% (in the eluate) for the first and second bsAbs, respectively. While more case studies are required to further demonstrate FcXP's superiority in aggregate removal, findings from the current study suggest that FcXP can potentially be a better alternative than Protein A for product capture in cases where aggregate content is high.

SEC-HPLC analysis of column load and flow-through provides critical understanding of low Protein A step yield.

For a certain number of mAbs, bispecific antibodies (bsAbs) and Fc-fusion proteins that we worked on, the Protein A capture step experienced low yield (i.e., ∼80%). A previous case study suggested that non-binding aggregate formed in cell culture was the root cause of low Protein A step yield. In the current work, we selected five projects with the low Protein A yield issue to further illustrate this phenomenon. In all cases, existence of non-binding aggregates was confirmed by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis of Protein A load and flow-through. In addition, we demonstrated that aggregates failed to bind to Protein A resin mainly due to their large sizes, which prevented them from entering the resin beads. As the data suggested, SEC-HPLC analysis of Protein A load and flow-through, although not a standard procedure, can provide information that is critical for understanding the unexpected performance of Protein A chromatography in cases like those being presented here. Thus, SEC-HPLC analysis of Protein A load and flow-through is highly recommended for antibodies/Fc-fusions suffering from low Protein A yield.

Impact of fed-batch process intensification on the productivity and product quality of two CHO cell lines expressing unique novel molecular format proteins.

While monospecific antibodies have long been the foundational offering of protein therapeutics, recent advancements in antibody engineering have allowed for the development of far more complex antibody structures. Novel molecular format (NMF) proteins, such as bispecific antibodies (BsAbs), are structures capable of multispecific binding, allowing for expanded therapeutic functionality. As demand for NMF proteins continues to rise, biomanufacturers face the challenge of increasing bioreactor process productivity while simultaneously maintaining consistent product quality. This challenge is exacerbated when producing structurally complex proteins with asymmetric modalities, as seen in NMFs. In this study, the impact of a high inoculation density (HID) fed-batch process on the productivity and product quality attributes of two CHO cell lines expressing unique NMFs, a monospecific antibody with an Fc-fusion protein and a bispecific antibody, compared to low inoculation density (LID) platform fed-batch processes was evaluated. It was observed that an intensified platform fed-batch process increased product concentrations by 33 and 109% for the two uniquely structured complex proteins in a shorter culture duration while maintaining similar product quality attributes to traditional fed-batch processes.

A non-genetic engineering platform for rapidly generating and expanding cancer-specific armed T cells.

BACKGROUND: Cancer-specific adoptive T cell therapy has achieved successful milestones in multiple clinical treatments. However, the commercial production of cancer-specific T cells is often hampered by laborious cell culture procedures, the concern of retrovirus-based gene transfection, or insufficient T cell purity. METHODS: In this study, we developed a non-genetic engineering technology for rapidly manufacturing a large amount of cancer-specific T cells by utilizing a unique anti-cancer/anti-CD3 bispecific antibody (BsAb) to directly culture human peripheral blood mononuclear cells (PBMCs). The anti-CD3 moiety of the BsAb bound to the T cell surface and stimulated the differentiation and proliferation of T cells in PBMCs. The anti-cancer moiety of the BsAb provided these BsAb-armed T cells with the cancer-targeting ability, which transformed the naïve T cells into cancer-specific BsAb-armed T cells. RESULTS: With this technology, a large amount of cancer-specific BsAb-armed T cells can be rapidly generated with a purity of over 90% in 7 days. These BsAb-armed T cells efficiently accumulated at the tumor site both in vitro and in vivo. Cytotoxins (perforin and granzyme) and cytokines (TNF-α and IFN-γ) were dramatically released from the BsAb-armed T cells after engaging cancer cells, resulting in a remarkable anti-cancer efficacy. Notably, the BsAb-armed T cells did not cause obvious cytokine release syndrome or tissue toxicity in SCID mice bearing human tumors. CONCLUSIONS: Collectively, the BsAb-armed T cell technology represents a simple, time-saving, and highly safe method to generate highly pure cancer-specific effector T cells, thereby providing an affordable T cell immunotherapy to patients.

Antibody aggregation can lead to reduced Protein A binding capacity and low step yield.

Protein A affinity chromatography has been widely used for antibody capture and initial purification. In general, the yield of this step is relatively high (i.e., >90%). However, recently while purifying a bispecific antibody (bsAb) in appended IgG format, the Protein A capture step experienced low yield (i.e., ∼80%). It was found that the target bsAb started appearing in flow-through at a relatively low load density, suggesting that a portion of the expressed bsAb has compromised Protein A binding capability. Further studies indicated that the bsAb in flow-through was mainly in aggregate form. In addition, normal Protein A step yield was restored when the column was loaded with bsAb monomer. Thus, all the evidence pointed to the fact that aggregate with compromised binding capacity was the cause of low Protein A step yield in this case.

Complementary methods for monitoring hole-hole homodimer associated with a WuXiBody-based asymmetric bispecific antibody.

WuXiBody is a bispecific antibody (bsAb) platform developed by WuXi Biologics. Its key feature is the replacement of one parental antibody's CH1/CL region with the T-cell receptor (TCR) constant domain, which prevents mispairing between non-cognate heavy chain and light chain. In addition, heavy chain heterodimerization in asymmetric WuXiBody molecule is promoted by the knobs-into-holes (KiH) technology. Despite the great success of KiH strategy in improving heterodimer formation, homodimers (especially the hole-hole homodimer) can still be generated at low levels. In general, detection and monitoring of homodimers during KiH bsAb purification are challenging as homodimers share similar physicochemical properties with the target heterodimeric bsAb. Nevertheless, the unique design of WuXiBody allows homodimers to be effectively detected and monitored by multiple methods. In the current work, with an asymmetric WuXiBody case study, we demonstrated that hole-hole homodimer can be effectively monitored by six chromatography methods including hydrophobic interaction chromatography (HIC), reverse phase (RP), cation exchange (CEX), KappaSelect, CaptureSelect CH1-XL and Protein L.

Construction and stable gene expression of AGR2xPD1 bi-specific antibody that enhances attachment between T-Cells and lung tumor cells, suppress tumor cell migration and promoting CD8 expression in cytotoxic T-cells.

There has been a substantial and consistent rise in the number of clinical trials to develop advanced and potent bispecific antibodies (BsAb) over the past two decades with multiple targets to improve the efficacy or tissue specificity of monoclonal antibodies (mAb) treatment for diseases with multiple determining factors or widely-expressed targets. In this study, we designed and synthesized BsAb AGR2xPD1 targeting extracellular AGR2, a paracrine signal, and PD1, an immune checkpoint protein. Our design is intended to use AGR2 binding to guide PD1 targeting for AGR2+cancer. We used this construction to produce AGR2xPD1 BsAb by generating clonally selected stable 293F cell line with high expression. Applying this BsAb in a T cell-Tumor cell co-culture system showed that targeting both PD1 and AGR2 with this BsAb induces the attachment of TALL-104 (CD8+ T-lymphocytes) cells onto co-cultured H460 AGR2+ Lung tumor cells and significantly reduces migration of H460 cells. T-cell expression of CD8 and IFNγ is also synergistically enhanced by the AGR2xPD1 BsAb treatment in the AGR2+H460 co-culture system. These effects are significantly reduced with AGR2 expression negative WI38 cells. Our results demonstrate that the AGR2xPD1 BsAb could be a potential therapeutic agent to provide better solid tumor targeting and synergetic efficacy for treating AGR2+ cancer by blocking AGR2 paracrine signaling to reduce tumor survival, and redirecting cytotoxic T-cells into AGR2+ cancer cells.

Advances in the study of CD47-based bispecific antibody in cancer immunotherapy.

Tumour therapy has entered the era of immunotherapy. Monoclonal antibodies (mAb), immune checkpoint inhibitors, chimeric antigen receptor T-cell (CAR-T), cytokine-induced killer (CIK), tumour-infiltrating lymphocytes (TILs) and other cellular immunotherapies have become the focus of current research. The CD47/SIRPα target is becoming another popular tumour immunotherapy target following the PDCD1/CD247(PD1/PD-L1) checkpoint inhibitor. In recent years, a large number of CD47/SIRPα mAbs, fusion proteins, and CD47/SIRPα-based bispecific antibodies (BsAbs) are undergoing preclinical and clinical trials and have good curative effects in the treatment of haematological tumours and solid tumours. They bring new vitality and hope for the treatment of patients with advanced tumours. This review summarizes the research progress of CD47/SIRPα-based BsAbs with different targets for tumour treatment. There are 12 and 9 BsAbs in clinical trials and pre-clinical research, respectively. We report on the mechanism of 15 BsAb molecules with different target and analyse the efficacy and safety of preclinical and clinical trials, discuss the issues that may be faced in the development of CD47-based BsAbs, and dual-target molecules, and summarize their development prospects. This review provides a reference for the safety and effectiveness of BsAbs in clinical application and in the future development of antibodies.

Prodrug-Activating Chain Exchange (PACE) converts targeted prodrug derivatives to functional bi- or multispecific antibodies.

Driven by the potential to broaden the target space of conventional monospecific antibodies, the field of multi-specific antibody derivatives is growing rapidly. The production and screening of these artificial proteins entails a high combinatorial complexity. Antibody-domain exchange was previously shown to be a versatile strategy to produce bispecific antibodies in a robust and efficient manner. Here, we show that the domain exchange reaction to generate hybrid antibodies also functions under physiological conditions. Accordingly, we modified the exchange partners for use in therapeutic applications, in which two inactive prodrugs convert into a product with additional functionalities. We exemplarily show the feasibility for generating active T cell bispecific antibodies from two inactive prodrugs, which per se do not activate T cells alone. The two complementary prodrugs harbor antigen-targeting Fabs and non-functional anti-CD3 Fvs fused to IgG-CH3 domains engineered to drive chain-exchange reactions between them. Importantly, Prodrug-Activating Chain Exchange (PACE) could be an attractive option to conditionally activate therapeutics at the target site. Several examples are provided that demonstrate the efficacy of PACE as a new principle of cancer immunotherapy in vitro and in a human xenograft model.

Targeting macrophages: a novel treatment strategy in solid tumors.

In the tumor microenvironment (TME), tumor-associated macrophages (TAMs) are the most abundant immune cells, which act as a key regulator in tumorigenesis and progression. Increasing evidence have demonstrated that the TME alters the nature of macrophages to maintain dynamic tissue homeostasis, allowing TAMs to acquire the ability to stimulate angiogenesis, promote tumor metastasis and recurrence, and suppress anti-tumor immune responses. Furthermore, tumors with high TAM infiltration have poor prognoses and are resistant to treatment. In the field of solid tumor, the exploration of tumor-promoting mechanisms of TAMs has attracted much attention and targeting TAMs has emerged as a promising immunotherapeutic strategy. Currently, the most common therapeutic options for targeting TAMs are as follows: the deletion of TAMs, the inhibition of TAMs recruitment, the release of phagocytosis by TAMs, and the reprogramming of macrophages to remodel their anti-tumor capacity. Promisingly, the study of chimeric antigen receptor macrophages (CAR-Ms) may provide even greater benefit for patients with solid tumors. In this review, we discuss how TAMs promote the progression of solid tumors as well as summarize emerging immunotherapeutic strategies that targeting macrophages.

Assessing four subdomain-specific affinity resins' capability to separate half-antibody from intact bispecific antibody.

Half-antibody is a frequent byproduct associated with the recombinant production of many asymmetric bispecific antibodies (bsAbs). Although this byproduct can be largely removed by post-capture polishing steps, it is ideal to have it partially cleared at the capture step to achieve a more robust downstream process. Previously we showed that Protein A affinity chromatography possesses the capability to separate half-antibody. In this study, we assessed the half-antibody separation capability of four less commonly used subdomain-specific affinity resins. The data suggest that these resins exhibit different capabilities for separating half-antibody from the corresponding bsAb. In specific, whereas Protein A affinity resin can always provide partial separation under typical conditions, the separation efficiency of three subdomain-specific affinity resins (i.e., Capto L, CaptureSelect CH1-XL and CaptureSelect FcXP) heavily relies on the property of the two parental monospecific antibodies from which the bsAb is derived, which may range from complete separation to no separation at all. This novel information provides more options for half-antibody clearance at the capture step.

Mechanism of action of a T cell-dependent bispecific antibody as a breakthrough immunotherapy against refractory colorectal cancer with an oncogenic mutation.

T cell-dependent bispecific antibody (TDB)-induced T cell activation, which can eliminate tumor cells independent of MHC engagement, is expected to be a novel breakthrough immunotherapy against refractory cancer. However, the mechanism of action of TDBs has not been fully elucidated thus far. We focused on TDB-induced T cell-tumor cell contact as an important initial step in direct T cell-mediated tumor cell killing via transport of cytotoxic cell proteases (e.g., granzymes) with or without immunological synapse formation. Using an anti-EGFR/CD3 TDB, hEx3, we visualized and quantified T cell-tumor cell contact and demonstrated a correlation between the degree of cell contact and TDB efficacy. We also found that cytokines, including interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα) secreted by activated T cells, damaged tumor cells in a cell contact-independent manner. Moreover, therapeutic experiences clearly indicated that hEx3, unlike conventional anti-EGFR antibodies, was effective against colorectal cancer (CRC) cells with mutant KRAS, BRAF, or PIK3CA. In a pharmacokinetic analysis, T cells spread gradually in accordance with the hEx3 distribution within tumor tissue. Accordingly, we propose that TDBs should have four action steps: 1st, passive targeting via size-dependent tumor accumulation; 2nd, active targeting via specific binding to tumor cells; 3rd, T cell redirection toward tumor cells; and 4th, TDB-induced cell contact-dependent (direct) or -independent (indirect) tumor cell killing. Finally, our TDB hEx3 may be a promising reagent against refractory CRC with an oncogenic mutation associated with a poor prognosis.

Comparing the relative robustness of byproduct removal by wash and by elution in column chromatography for the purification of a bispecific antibody.

For recombinant antibody purification, removal of product-related impurities usually relies on the two polishing steps post Protein A chromatography. A certain impurity may bind weaker or tighter to a particular type of column than the target antibody, and this forms the basis for separation. For impurities that bind weaker, they can be removed by pre-elution wash under appropriate conditions. For impurities that bind stronger, they can be separated by using a suitable condition that selectively elutes the product. In this study, with a bispecific antibody case, we compared the relative robustness of byproduct removal by wash and by elution using two different types of chromatography. The data suggest that elution-enabled byproduct clearance is more robust than wash-enabled clearance, and the former approach provides consistent impurity clearance over a relatively wide range of loading density.

Evaluating a new mixed-mode resin Diamond MMC Mustang using Capto MMC ImpRes as a benchmark.

Diamond MMC Mustang is a relatively new mixed-mode resin, which mediates both cation exchange and hydrophobic interactions. In this work, we evaluated this resin using Cytiva's Capto MMC ImpRes, a well-established mixed-mode resin with similar properties, as a benchmark. The data suggest that in comparison with Capto MMC ImpRes, Diamond MMC Mustang exhibits comparable binding capacity and resolution. In addition, the resin under evaluation shows good lot-to-lot consistency. The information provided in this study allows users to have additional options when selecting mixed-mode resin for intermediate purification or final polishing, which is favourable especially at the present time when the supply chains of many manufacturers are negatively impacted by the coronavirus pandemic.

Impact of salt concentration in mobile phase on antibody retention in Protein A, Protein L and KappaSelect affinity chromatography.

Protein A, Protein L and KappaSelect affinity resins have been widely used for antibody purification. Elution of antibody bound to these resins is typically achieved by acidic pH. In addition, elution can be moderately adjusted by tuning the salt concentration in mobile phase as hydrophobic interactions play a major role in binding. In this study, we assessed the impact of salt concentration in mobile phase on antibody retention in these three types of affinity chromatography. The data suggest that salt concentration has a bigger impact on retention in the two light chain-binding affinity columns (i.e., Protein L and KappaSelect) than in Protein A column. In particular, lowering salt concentration in mobile phase for Protein L and KappaSelect columns allows elution become feasible at higher pH. In addition, this finding suggests that wash in these two types of column aimed at removing weakly-bound byproducts can also be performed at increased pH by lowering salt concentration in the wash buffer. Rendering wash and elution feasible at higher pH has practical value for cases where the target antibodies are sensitive to stringent conditions.

Removing a single-arm species by Fibro PrismA in purifying an asymmetric IgG-like bispecific antibody.

MabSelect PrismA is an affinity resin whose ligand is derived from the B-domain of Protein A. Compared to its predecessor resins (i.e., MabSelect SuRe LX and MabSelect SuRe), the PrismA ligand has enhanced affinity for the VH3 chain. Consistently, when being used for the purification of an asymmetric IgG-like bispecific antibody (bsAb), MabSelect PrismA resin shows certain resolution between the intact product and a single-arm byproduct, which contain the same Fc region but different numbers of VH3 domain. Fibro PrismA is a newly launched product which has the same PrismA Protein A ligand as MabSelect PrismA but uses cellulose fiber instead of resin as its matrix. It was learned that Fibro PrismA, in comparison to PrismA resin, exhibits further improved resolution, allowing efficient clearance of the single-arm byproduct as well as good recovery of the target bsAb. This finding suggests that Fibro PrismA provides a potential solution for separating antibody species containing the same Fc region but different numbers of VH3 domain, which can otherwise be difficult to separate.

Immunoglobulin-binding protein-based affinity chromatography in bispecific antibody purification: Functions beyond product capture.

In general, purification of bispecific antibody (bsAb) is more challenging than that of monospecific antibody due to the increased complexity in byproduct profile. Like in the case of monospecific antibody purification, immunoglobulin-binding protein-based affinity chromatography is an indispensable tool for bsAb purification. For example, Protein A affinity chromatography has been widely used to capture Fc-containing bsAbs whereas other affinity media such as Protein L and KappaSelect, which bind kappa light chain, are used to capture bsAbs that do not contain a Protein A-binding site. In fact, affinity chromatography also possesses the capability of removing certain product-related impurities in bsAb purification when it is conducted with suitable medium and under appropriate conditions. Fully exploring the potential of affinity chromatography in bsAb purification to achieve both product capture and byproduct removal is highly desirable, as this can greatly alleviate the purification burden on subsequent polishing steps and hence improves the overall robustness of the downstream process. This article briefly reviews the byproduct clearance potential of several commonly used affinity media under relevant bsAb purification scenarios.

Application of pH-salt dual gradient elution in purifying a WuXiBody-based bispecific antibody by MMC ImpRes mixed-mode chromatography.

WuXiBody is a novel bispecific antibody (bsAb) platform developed by WuXi Biologics. BsAbs based on WuXiBody can adopt either asymmetric or symmetric format. In general, recombinant production of asymmetric bsAbs has a more complex impurity profile than that of symmetric ones, as assembly of the former usually involves an increased number of distinct chains. Consequently, purification of asymmetric bsAb typically poses greater challenges to the downstream team. Recently while purifying a WuXiBody-based bsAb in asymmetric format, we learned that MMC ImpRes mixed-mode chromatography under linear pH or salt gradient elution can effectively remove two out of the three low-molecular-weight byproducts post Protein A chromatography but not the third one, which was eventually removed under pH-salt dual gradient elution. This finding suggests that MMC ImpRes chromatography performed under pH-salt dual gradient elution provides better resolution than that performed under pH/salt mono gradient elution.