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Targeting two receptors can significantly increase cell specificity

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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 Dr. Jonathan Davis. The summary was kindly written by Dr. Czeslaw Radziejewski.

 


Targeting two receptors can significantly increase cell specificity.
Jonathan Davis, Vice President of Innovation and Strategy, Invenra, Inc.

Jonathan Davis presented a talk detailing Invenra’s rationale for generating bispecific antibodies that target two receptors at the same cell and provided some examples of their biological activity. The platform is based on the construct in which CH1/CL domain in one arm is substituted with a domain derived from CH3. This approach produces stable constructs that are easy to purify. The presentation focused on bispecifics referred to as SNIPERs. The idea behind bispecific SNIPERs is to combine two binding arms, both of which having low affinity toward their cellular targets. When both targets are engaged with cognate targets on the cell surface, the avidity effect results in much stronger binding. This approach could potentially address undesirable binding of monospecific antibodies to healthy tissues where tumor antigen is also expressed at lower levels.

Dr. Davis discussed the concept of symmetric synergy and asymmetric synergy. In the case of symmetric synergy both targets are present at about the same density, whereas in asymmetric synergy one target is present in much greater abundance than the other. According to the speaker, for the symmetric synergy to occur the two target molecules have to be in a right orientation, so the epitopes have to be properly oriented in respect to each other, at least most of the time. This necessitates screening large number of antibodies in order to build a bispecific that demonstrates good synergy. With good geometry fit, 100- to 1000-fold increases in affinity can be reached on cells. He cited the IL-2 receptor system as an example of asymmetric synergy found in nature. High affinity IL-2 receptor is a three-part system consisting of alpha, beta, and gamma subunits. The alpha subunit is present in high concentration, but binds IL-2 with low affinity. The alpha subunit with bound IL-2 binds to beta and then to gamma subunits to form a high affinity signaling complex. This process goes in one direction: from alpha to beta and gamma and that is why it is considered asymmetric. Dr. Davis emphasized that Invenra has the ability to generate and screen large number of constructs to select the right candidate for further development.

Invenra is exploring the SNIPER approach for Treg depletion and for the agonism of co-stimulatory receptor for T cells, OX40. In this lecture, Dr. Davis discussed the anti-tumor activity of SNIPER INV721 in neuroblastoma. The marketed antibody therapeutic, dinutuximab, targets disialoganglioside GD2 that is densely expressed on neuroblastoma cells. GD2 is also expressed on melanomas, small cell lung cancers and sarcomas. Dinutuximab causes lysis of GD2-expressing cells and its mechanism of action involves ADCC and CDC. The antibody is very effective, but causes excruciating pain in patients, presumably because the ganglioside is expressed in all tissues, albeit at the much lower levels. As a second target of INV721, Invenra selected the check-point molecule B7H3 (CD276) that is present only on the tumor cells. To reduce affinity for ganglioside GD2, some residues in the existing antibody against the target were mutated, which allowed the generation of SNIPER( INV721) that bound to neuroblastoma cells only if two targets were present, but not either one alone. To test the in vivo binding affinity of the bispecific antibody, INV721 was radiolabeled with 89Zr. Mice bearing GD2/B7H3-expressing tumors were intravenously injected with 89Zr-labeled INV721 and its in vivo biodistribution was monitored via positron emission tomography imaging. 89Zr-INV721- showed elevated accumulation in the tumor with minimal uptake in normal tissues. 89Zr-radiolabeled isotype control antibody displayed significantly lower tumor uptake demonstrating the specificity of INV721. (1) Dr. Davis indicated that one potential extension of the Invenra bispecific antibodies approach would be to convert these molecules into T-cell engagers.

1. Erbe AK et al. Specific Targeting of Tumors Through Bispecific SNIPER Antibodies. J Immunol, May 1, 2020, 204 (1 Supplement) 91.2.

Join us on April 7th for our next webinar, Precision Execution of Bispecifics at Scale from Design to Delivery!

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Thursday April 7, 2022 11am ET / 5pm CET
Speaker: Dr. Lisa Prendergast, Associate Director of Expression System Sciences in Licensing at Lonza

Registration for our next webinar, “Precision Execution of Bispecifics at Scale from Design to Delivery“, is now open!

Novel therapeutic modalities such as bispecific antibodies are increasingly being explored as more effective alternatives to monoclonal antibodies for a range of diseases. Therapeutics such as bispecifics, can have a combinatorial effect by targeting two antigens,  resulting in treatments with enhanced utility, higher efficacy, fewer side effects and less resistance compared to mAbs.

Generating a bispecific antibody, which is correctly and stably paired, is a major production concern. Many solutions require significant changes to native antibody structure, which increases antibody complexity and forces adaptation of downstream processes. While a various platforms have been developed to mitigate Heavy-Light chain (HC-LC) mispairing, there are many other rate limiting steps for efficiently expressing these molecules in a CHO system. bYlok® technology is a design engineering approach that stabilise the interaction between the HC and LC, essentially removing the mispairing problem whilst retaining a more natural antibody structure.

This presentation will introduce you to a mechanistic review of the bispecific pipeline to demonstrate how a various tools and technologies can enable you execute bispecifics.  Case studies will be presented to show how the bYlok® technology can be used to stabilise and select for novel bispecifics from a panel of parental immunotherapeutic mAbs. Our data demonstrates that correct heterodimerisation can be achieved consistently and how standard downstream purification processes can be used during production.

Register here!

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

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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.

Discovering and Targeting Neo-epitopes in Cancer

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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 plenary lecture by Prof. James Wells (USCF), kindly written by Dr. Czeslaw Radziejewski.

 


Discovering and Targeting Neo-epitopes in Cancer.
James Wells, Professor and Chair, Department of Pharmaceutical Chemistry, UCSF

Professor Wells presented the plenary lecture on the identification of cancer-associated proteolytic neo-epitopes in cell membrane proteins and the identification of novel cancer-specific MHC-1 peptide complexes. Cell surface proteins are the targets of most biologic and small molecule drugs. Professor Wells and colleagues use cell surface proteomics to examine changes in the cell surface proteins upon transformation with oncogenes such as KRAS, HER2, EGFR, BRAF, MEK, and Myc. Ecto-domains of identified proteins, which generally belong to the single pass trans-membrane class, are expressed as Fc fusion proteins and antibodies are generated against these proteins via screening phage libraries. Specificities of the antibodies are verified by testing against full-length trans-membrane proteins expressed by cells transfected with appropriate vectors.

Proteolysis is a primary post-translational modification of cell surface proteins. There are approximately 500 human proteases, and proteolysis plays an important role in disease progression, such as angiogenesis, invasion and metastasis, inflammation, and immune evasion. Well’s lab is exploring methods to identify proteolytic cleavage sites on the surfaceome of cancer cells.[1] To accomplish this, they devised a technology called N-terminomics, which uses the peptide ligase called subtiligase. Subtiligase ligates peptide esters to the N-terminus of a protein or a peptide. This enzyme can be used for other purposes, such as peptide cyclization and protein bioconjugation. The lab used peptides tagged with biotin or fluorescently labelled in conjunction with mass spectrometry to identify sites of proteolytic cleavage.[2,3] Prof. Wells showed an example of this strategy used to identify sites of cleavage by caspase in the proteome of a human cell line in which apoptosis was induced. This approach, however, identified only a limited number of cleaved proteins. In the next implementation of the strategy, cells were directly transfected with subtiligase. This strategy allowed the identification of hundreds of extracellular proteins that were proteolytically modified.

The newest strategy invented in Prof. Wells’ lab (unpublished) involves tethering subtiligase to glycans of cell surface proteins instead of transacting cells. Using this latest strategy in Kras-transformed cells, 611 cell surface cleavage events were observed. In HER2-transfected cells, 267 cleavage events were observed and the majority of events were not related to cleavage of signal peptide from extracellular proteins. Interestingly, the extent of proteolytic modification of some proteins in oncogene-transformed cells can either increase or decrease. Similarly, expression levels of the same proteins also change in both directions. N-terminomics of Kras- and HER2-transformed cells was thus different.

This study also identified an interesting protein called CDCP1, which has cleavage and expression that is upregulated in pancreatic cancer. The cleavage is indeed specific to cancer cells. Three closely nested cleavage sites were found in CDCP1. Antibodies (CL03.2) were developed in the lab against the cleaved form  of CDCP1. Cells containing the cleaved form were efficiently killed by the anti-CDCP1 antibody formatted as an antibody-drug conjugate (ADC). In Jurkat cells, an anti-CD3/anti-CDCP1 bispecific single-chain variable fragment showed killing activity. For in vivo studies, mouse-specific antibodies toward the truncated form of CDCP1 were generated and used to produce an auristatin (MMAF)-based ADC. An ADC against the truncated form of CDCp1 was well tolerated in non-tumor-bearing mouse, but the animals lost weight when treated with an ADC targeting the full-length protein. In a study of mice bearing xenograph tumors, the animals were administered antibody against the truncated form that was radiolabeled with isotope Lu 177 and a dramatic decrease of tumor growth was observed.

[Read more…]

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

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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.