The Antibody Society

the official website of the antibody society

An international non-profit supporting antibody-related research and development.

You are here: Home / Archives for Bispecific antibodies

Clinical-stage ROR1xCD3 bispecific antibodies with potential for broad cancer specificity

by

Antibody Engineering & Therapeutics, held in December 2021, offered many opportunities to hear exciting and informative presentations by experts in the field. We are pleased to present here a summary of a lecture given in the “Immune Cell Recruitment and Redirection” session by Prof. Kerry Chester. The summary was kindly written by Dr. Czeslaw Radziejewski.

Clinical-stage ROR1xCD3 bispecific antibodies with potential for broad cancer specificity.
Kerry Chester, Professor of Molecular Medicine at University College London and CSO of Novalgen.

The leading molecule of Novalgen is NVG-111, a first-in-class tandem T-cell engager in single-chain variable fragment (scFv) format. One arm of NVG-111 targets a T-cell coreceptor, CD3, while the second binds to the tumor-associated tyrosine kinase-like receptor ROR1. ROR1 was cloned in 1992 from a neuroblastoma cell line. (1) The function of ROR1 as a tyrosine kinase is still poorly understood, although some studies show evidence of its intrinsic tyrosine kinase activity. ROR1 is a cell-surface oncofetal antigen, expressed during embryogenesis and largely absent in normal adult organs, with only low-level expression on adipocytes, pancreas, and parathyroid glands. In contrast to the lack of expression in healthy tissues, ROR1 is present in a wide range of cancers and cancer initiating stem cells. It is expressed in both hematological malignancies and in solid tumors. (2)

ROR1 has three extracellular domains: Kringle, Frizzled and Ig-like domain. ROR1 sequences of extracellular domain (ECD) are highly similar between different species. For example, there is 97.6% identity between mouse and human ROR1 ECD. Many years after the initial ROR1 discovery, its ligand was identified as Wnt-5a, one of the Wnt family signaling molecules. Unlike other ROR1 clinical candidates under development, the anti-ROR1 arm of NVG-111 binds to ROR1 Frizzled domain.

Novalgen began the development of NVG-111 by immunizing rats with recombinant extracellular domain of ROR1. The majority of the resulting antibodies bound to Ig-like domain, none bound to Kringle domain, and only one clone (clone F) bound to Frizzled domain. Clone F was selected for further development. Using flow-cytometry, Novalgen demonstrated binding of clone F to a large number of human cancer cell lines. Clone F was humanized and used to format a bispecific scFv with humanized anti-CD3. NVG-111 binds to mouse and to human ROR1 with low nanomolar affinity, but the anti-CD3 arm does not bind to mouse CD3.

In preclinical studies NVG-111 was effective in in-vitro and in an in-vivo mice model of hematological malignancies, and it demonstrated the ability to kill solid tumor in an established PANC-1 mouse xenograft model of human pancreatic carcinoma. NVG-111 also demonstrated killing in models of advanced solid tumors. It eliminated CD44+/CD24- cancer stem cells in a solid tumor model of triple-negative breast cancer. It induced dose-dependent killing in chronic lymphocytic leukemia (CLL) patient samples where patient CLL cells were cocultured with autologous T cells with EC50 in the range of 4-100 pg/ml. NVG-111 showed T cell-mediated killing of mantle cell lymphoma (MCL) cells that was as effective as killing by blinatumomab, which binds CD3 and CD19, but with 2—30% lower levels of cytokine release (measured as interferon gamma) than blinatumomab, suggesting lower risk of cytokine-release syndrome. Toxicity studies performed in mice using AAV expressing NVG-111 showed lack of toxicity at levels 20- to 1000-fold of expected steady-state levels in clinical dose. Because over 90% of CLL/MCL patients are ROR1 positive, the current focus of Novalgen clinical studies are these two hematological malignancies. Importantly, ROR1 is not expressed on normal B cells, therefore risk of B cell aplasia is expected to be reduced.

1. Masiakowski P, Carroll RD. A novel family of cell surface receptors with tyrosine kinase-like domain. J Biol Chem. 1992;267(36):26181-90.

2. Yuming Zhao et al. Tyrosine kinase ROR1 as a target for anti-cancer therapies. Front. Oncol., 11:680834. doi: 10.3389/fonc.2021.680834.

FDA approves Vabysmo™ (faricimab-svoa) for ophthalmic disorders

by

On January 28, 2022, Genentech announced that the U.S. Food and Drug Administration has approved Vabysmo ™ (faricimab-svoa) for the treatment of wet, or neovascular, age-related macular degeneration (AMD) and diabetic macular edema (DME). Faricimab (RO6867461, RG7716) is an anti-vascular endothelial growth factor-A (VEGF-A) and anti-angiopoietin-2 (Ang-2) bispecific antibody derived from Roche’s CrossMab technology.

The approval was based in part on results from four Phase 3 studies in wet AMD and DME. The randomized, double-masked, and active comparator-controlled TENAYA (NCT03823287) and LUCERNE (NCT03823300) studies evaluated the effects of faricimab (6.0 mg administered at fixed intervals of every two, three, or four months) and aflibercept (Eylea®) (2.0 mg administered at fixed two-month intervals) in wet AMD patients. The primary endpoint of the studies, average change in best-corrected visual acuity (BCVA) from baseline through week 48, was met in both studies. The average vision gains from baseline in the faricimab arms were +5.8 and +6.6 letters, compared to +5.1 and +6.6 letters in the aflibercept arms, in the TENAYA and LUCERNE studies, respectively, demonstrating the non-inferiority of faricimab compared to aflibercept. The study also showed that faricimab’s treatment interval could be longer than that of aflibercept – nearly 80% of patients treated with faricimab were able to go three months or longer between treatments during the first year.

The 3-arm, randomized, double-masked, active comparator-controlled YOSEMITE (NCT03622580) and RHINE studies (NCT03622593) compared the effects of faricimab (6.0 mg administered at personalized treatment intervals (PTI) of up to four months or 6.0 mg administered at fixed two-month intervals) to those of aflibercept (2.0 mg administered at fixed two-month intervals) in DME patients. The primary endpoint, average change in BCVA score from baseline at one year, was met, with faricimab again showing non-inferiority in visual acuity gains compared to aflibercept. In the YOSEMITE study, the average vision gains from baseline were +11.6, +10.7, and +10.9 letters eye chart letters in the faricimab PTI, faricimab two-month, and aflibercept arms, respectively. The average vision gains from baseline were +10.8, +11.8, and +10.3 letters in the faricimab PTI, faricimab two-month, and aflibercept arms, respectively, in the RHINE study.

Further details for the TENAYA and LUCERNE and YOSEMITE and RHINE studies were published in The Lancet.

The European Medicines Agency has accepted the submission of a Marketing Authorisation Application for faricimab.

Vabysmo ™ is the 2nd antibody-based therapeutic granted a first approval for marketing in the EU or US in 2022. Explore our searchable table of antibody therapeutics approved in the EU or US for details.

Bispecific antibodies come to the fore

by

Bispecific antibodies are a versatile class of targeted therapeutics designed to bind two different sites, which can be located on a single antigen or on two antigens. Although bispecific antibodies were conceptualized ~60 years ago, various challenges associated with protein engineering, stability and manufacturing delayed their wide-spread development. However, as of 2020, numerous validated platforms, i.e., those that have produced bispecific clinical candidates, are readily available (1). Using these platforms, the commercial clinical pipeline has grown to over 100 bispecific antibodies, ranging from tandem single-chain variable fragments (scFv) to full-length immunoglobulins with dual variable domains. Substantial growth in the pipeline has occurred only relatively recently, though. During the early 2010s, bispecific antibodies comprised less than 10% of the total number of antibody therapeutics entering clinical study per year, but this number rose to 25% by 2018. Reflecting the general success of antibody therapeutics, the entry of all types of new, innovative antibody candidates into clinical study also grew substantially during this period, from 63 on average during the early 2010s to over 140 in 2018.

As is the case for the overall pipeline of antibody therapeutics, the majority of bispecific antibodies that have entered clinical study recently are being evaluated as treatments for cancer. Among these, the most common approach involves guiding T cells to cancer cells via a bispecific antibody, which binds to a tumor-associated antigen on a cancer cell and CD3 on T cells. Bispecifics that use this mechanism of action comprise ~45% of the pipeline. Of the T-cell engaging bispecifics now in the clinic, B-cell maturation antigen is the tumor-associated antigen most frequently targeted, followed by CD20, CD33, CD123 and prostate-specific membrane antigen. Of the bispecific antibodies in the clinical pipeline that do not re-direct T cells, the most frequent targets are programmed cell death 1 (PD1) and its ligand (PD-L1), human epidermal growth factor 2 (HER2) and vascular endothelial growth factor (VEGF). The most frequently paired targets are HER2/HER2 (different epitopes), PD1/CTLA4, PD-L1/4-1BB, VEGF/Ang-2 and VEGF/Delta-like ligand 4. Immune checkpoint proteins are frequent targets, including PD1 paired with LAG3, ICOS and TIM3, as well as PD-L1 paired with LAG3 and CTLA4.

The increased number of antibody therapeutics in the commercial clinical pipeline is due, at least in part, to the relatively high approval success rate of these molecules. Since 2014, at least 6 antibody therapeutics have been approved in either the US or European Union each year, and the number of approvals in 2020 is expected to exceed that of the all-time high of 13 approvals set in 2018 (2). Overall, antibody therapeutics have a 22% approval success rate, defined as the percentage of molecules that successfully transitioned from Phase 1 to approval of all that entered Phase 1 (3). For each clinical phase transition, the lowest rates are for the transition from Phase 1 to 2 (69%) and from Phase 2 to 3 (45%). So far, bispecific antibodies are very similar to the broader category of antibody therapeutics in their Phase 1 to 2 (71%) and Phase 2 to 3 (46%) transition rates. Since so few bispecific antibodies have reached Phase 3 or been approved, there is insufficient data for the calculation of meaningful transition rates for Phase 3 to regulatory review and regulatory review to approval. Despite this, the favorable early phase transition rates are good news for bispecific antibody developers.

In addition to success rates, the length of time required for clinical development and regulatory review is a key drug development metric. Typically for antibody therapeutics, 4-6 years is considered a relatively short period, ~ 8 years is about average, and a period of 10-12 years is considered lengthy. As with success rates, a meaningful average development period for bispecific antibodies is not available because only 3 have been approved (emicizumab, catumaxomab, blinatumomab), and 2 of these are likely not representative of bispecifics currently in clinical development. Of the 3 approved products, emicizumab, a humanized IgG4 targeting Factor IXa and Factor X approved for hemophilia, proceeded through clinical development to approval the fastest (~5.25 years), and it is most similar in structure to a canonical IgG antibody. In contrast, blinatumomab took the longest (~13 years), and it is the most dissimilar to a canonical IgG, which is typically includes human or humanized protein sequence. Blinatumomab is a tandem scFv composed of murine protein sequence with such a short half-life (2.1 hours) that continuous intravenous dosing is required for efficacy.

Because most bispecific antibodies in the commercial pipeline entered clinical studies in just the past few years, marketing approvals, if granted, may not occur for at least 4-5 years. However, two bispecific antibodies, tebentafusp and faricimab, qualify as ‘Antibodies to Watch’ (2) with late-stage clinical study primary completion dates in 2020. Tebentafusp, which is composed of a soluble T cell receptor fused to an anti-CD3 scFv (4), is being evaluated in a pivotal Phase 2 study with a primary completion date in July 2020. Faricimab is a bispecific CrossMAb (5) targeting VEGF-A and Ang-2 undergoing evaluation in several Phase 3 studies with primary completion dates in September 2020. Tebentafusp and faricimab are being studied as treatments for uveal melanoma and diabetic macular edema, respectively. Results from the clinical studies, which will help determine whether the molecules advance to regulatory review, may be available in the second half of 2020.

In summary, bispecific antibodies are entering clinical studies in record numbers, with most developed for cancer. Data available to date indicates that these molecules have similar early clinical phase transition rates, and the potential for similar development periods, compared with canonical IgG antibodies. Data discussed here will be updated and presented at PEGS Boston in the “Clinical Validation of Platforms” session of the “Engineering Bispecific Antibodies” track on Friday May 8, 2020.

1.      Labrijn AF, Janmaat ML, Reichert JM, Parren PWHI. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov. 2019;18(8):585–608. doi:10.1038/s41573-019-0028-1

2.      Kaplon H, Muralidharan M, Schneider Z, Reichert JM. Antibodies to watch in 2020. MAbs. 2020;12(1):1703531. doi:10.1080/19420862.2019.1703531

3.      Kaplon H, Reichert JM. Antibodies to watch in 2019. MAbs. 2019;11(2):219–238. doi:10.1080/19420862.2018.1556465

4.      Damato BE, Dukes J, Goodall H, Carvajal RD. Tebentafusp: T cell redirection for the treatment of metastatic uveal melanoma. Cancers (Basel). 2019;11(7):971. Published 2019 Jul 11. doi:10.3390/cancers11070971.

5.      Klein C, Schaefer W, Regula JT. The use of CrossMAb technology for the generation of bi- and multispecific antibodies [published correction appears in MAbs. 2018 Nov 13;11(1):217]. MAbs. 2016;8(6):1010–1020. doi:10.1080/19420862.2016.1197457

Most read from mAbs, Nov/Dec 2019

by

The Antibody Society is pleased to be affiliated with mAbs, a multi-disciplinary journal dedicated to advancing the art and science of antibody research and development. We hope you enjoy these summaries based on the abstracts of the most read papers published in a recent issue.

All the articles are open access; PDFs can be freely downloaded by following the links below.

Issue 11.8 (Nov/Dec 2019)

Insights into the IgG heavy chain engineering patent landscape as applied to IgG4 antibody development

In this new Perspective, Dumet et al., present the results from their study of the patent landscape of IgG4 Fc engineering, i.e., patents claiming modifications in the heavy chain. Thirty-seven relevant patent families were identified, comprising hundreds of IgG4 Fc variants focusing on removal of residual effector functions (since IgG4s bind to FcγRI and weakly to other FcγRs), half-life enhancement and IgG4 stability. Given the number of expired or soon to expire major patents in those 3 areas, companies developing blocking antibodies now have, or will in the near future, access to free tools to design silenced, half-life extended and stable IgG4 antibodies.

Antibody discovery and engineering by enhanced CRISPR-Cas9 integration of variable gene cassette libraries in mammalian cells

Parola et al. describe an antibody engineering and screening approach where complete variable light (VL) and heavy (VH) chain cassette libraries are stably integrated into the genome of hybridoma cells by enhanced Cas9-driven homology-directed repair (HDR), resulting in their surface display and secretion. By developing an improved HDR donor format that utilizes in situ linearization, they were able to achieve >15-fold improvement of genomic integration, resulting in a screening workflow that only requires a simple plasmid electroporation. This proved suitable for different applications in antibody discovery and engineering. By integrating and screening an immune library obtained from the variable gene repertoire of an immunized mouse, they isolated a diverse panel of >40 unique antigen-binding variants. They also successfully performed affinity maturation by directed evolution screening of an antibody library based on random mutagenesis, leading to the isolation of several clones with affinities in the picomolar range.

DuoMab: a novel CrossMab-based IgG-derived antibody format for enhanced antibody-dependent cell-mediated cytotoxicity

In this new Report,  Sustmann et al. present a generic approach to generate two novel IgG-derived antibody formats that are based on a modification of the CrossMab technology. MoAbs harbor two heavy chains (HCs) resulting in one binding entity and one Fc, whereas DuoMabs are composed of four HCs harboring two binding entities and two Fc regions linked at a disulfide-bridged hinge. The latter bivalent format is characterized by avidity-enhanced target cell binding while simultaneously increasing the ‘Fc-load’ on the surface. DuoMabs were shown to be producible in high yield and purity and bind to surface cells with affinities comparable to IgGs. The increased Fc load directed at the surface of target cells by DuoMabs modulates their ADCC competency toward target cells, making them attractive for applications that require or are modulated by FcR interactions.

Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development

Heavy chain (Hc) heterodimers represent a majority of bispecific antibodies (bsAbs) under clinical development. Although recent technologies achieve high levels of Hc heterodimerization (HD), traces of homodimer contaminants are often present, and as a consequence robust purification techniques for generating highly pure heterodimers in a single step are needed. Ollier et al. describe two different purification methods that exploit differences in Protein A (PA) or Protein G (PG) avidity between homo- and heterodimers. Differential elution between species was enabled by removing PA or PG binding in one of the Hcs of the bsAb. The PA method allowed the avidity purification of heterodimers based on the VH3 subclass, which naturally binds PA and interferes with separation, by using a combination of IgG3 Fc and a single amino acid change in VH3, N82aS. The PG method relied on a combination of three mutations that completely disrupts PG binding, M428G/N434A in IgG1 Fc and K213V in IgG1 CH1. Both methods achieved a high level of heterodimer purity as single-step techniques without Hc HD (93–98%). Since PA and PG have overlapping binding sites with the neonatal Fc receptor (FcRn), they investigated the effects of the engineering both in vitro and in vivo. Mild to moderate differences in FcRn binding and Fc thermal stability were observed, but these did not significantly change the serum half-lives of engineered control antibodies and heterodimers. The methods are conceptually compatible with various Hc HD platforms such as BEAT® (Bispecific Engagement by Antibodies based on the T cell receptor), in which the PA method has already been successfully implemented.

Most read from mAbs, May-June 2019

by

The Antibody Society is pleased  to be affiliated with mAbs, a multi-disciplinary journal dedicated to advancing the art and science of antibody research and development. We hope you enjoy these summaries based on the abstracts of the most read papers published in a recent issue.

All the articles are open access; PDFs can be freely downloaded by following the links below.

Issue 11.4 (May-June 2019)

Combining the best of two worlds: highly flexible chimeric antigen receptor adaptor molecules (CAR-adaptors) for the recruitment of chimeric antigen receptor T cells.

In this review, Darowski et al. summarize emerging approaches that aim to further evolve CAR-T cell therapy based on combinations of so-called universal or modular CAR-(modCAR-)T cells, and their respective adaptor molecules (CAR-adaptors), which mediate the crosslinking between target and effector cells. The activity of such modCAR-T cells is entirely dependent on binding of the respective CAR-adaptor to both a tumor antigen and to the CAR-expressing T cell. Contrary to conventional CAR-T cells, where the immunological synapse is established by direct interaction of CAR and membrane-bound target, modCAR-T cells provide a highly flexible and customizable development of the CAR-T cell concept and offer an additional possibility to control T cell activity.

Efficient tumor killing and minimal cytokine release with novel T-cell agonist bispecific antibodies.

Using a sequence-based discovery platform, Trinklein et al. identified new anti-CD3 antibodies from humanized rats that bind to multiple epitopes and elicit varying levels of T-cell activation. In T-BsAb format, 12 different anti-CD3 arms induce equivalent levels of tumor cell lysis by primary T-cells, but potency varies by a thousand-fold. The lead CD3-targeting arm stimulates very low levels of cytokine release, but drives robust tumor antigen-specific killing in vitro and in a mouse xenograft model. This new CD3-targeting antibody underpins a next-generation T-BsAb platform in which potent cytotoxicity is uncoupled from high levels of cytokine release, which may lead to a wider therapeutic window in the clinic.

Sym021, a promising anti-PD1 clinical candidate antibody derived from a new chicken antibody discovery platform.

In this study by Gjetting et al., the Symplex antibody discovery platform was adapted to chicken immunoglobulin genes and combined with high-throughput humanization of antibody frameworks by “mass complementarity-determining region grafting”. Wild type chickens were immunized with an immune checkpoint inhibitor programmed cell death 1 (PD1) antigen, and a repertoire of 144 antibodies was generated. The PD1 antibody repertoire was successfully humanized, and the authors found that most humanized antibodies retained affinity largely similar to that of the parental chicken antibodies. The lead antibody Sym021 blocked PD-L1 and PD-L2 ligand binding, resulting in elevated T-cell cytokine production in vitro. Detailed epitope mapping showed that the epitope recognized by Sym021 was unique compared to the clinically approved PD1 antibodies pembrolizumab and nivolumab. Moreover, Sym021 bound human PD1 with a stronger affinity (30 pM) compared to nivolumab and pembrolizumab, while also cross-reacting with cynomolgus and mouse PD1. This enabled direct testing of Sym021 in the syngeneic mouse in vivo cancer models and evaluation of preclinical toxicology in cynomolgus monkeys. Preclinical in vivo evaluation in various murine and human tumor models demonstrated a pronounced anti-tumor effect of Sym021, supporting its current evaluation in a Phase 1 clinical trial.