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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.

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.

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FDA issues a complete response letter for retifanlimab’s BLA

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On July 23, 2021, Incyte Corporation announced that the U.S. Food and Drug Administration (FDA) issued a Complete Response letter regarding its Biologics License Application (BLA) for retifanlimab (formerly INCMGA00012, MGA012) for the treatment of adult patients with locally advanced or metastatic squamous cell carcinoma of the anal canal (SCAC) who have progressed on, or who are intolerant of, platinum-based chemotherapy. Retifanlimab, which is a humanized, hinge-stabilized IgG4κ monoclonal antibody targeting programmed cell death protein 1 (PD-1), was granted FDA’s Fast track and Orphan Drug designations for the treatment of anal cancer.

The BLA submission was based on data from the Phase 2 POD1UM-202 trial (NCT03597295) evaluating retifanlimab in previously treated patients with locally advanced or metastatic SCAC who have progressed on, or were ineligible for or intolerant of, platinum-based chemotherapy. The objective response rate was 13.8% (95% confidence interval [Cl]: 7.6, 22.5) based on confirmed tumor responses by independent central radiographic review. Twelve patients (12.8%) had partial responses, 1 patient (1.1 %) had a complete response and 33 (35.1%) had stable disease. On June 24, 2021, FDA’s Oncologic Drug Advisory Committee had voted 13 to 4 for the deferral of the FDA approval of retifanlimab. FDA’s letter indicates that the application cannot be approved in its present form and additional data are needed to demonstrate the clinical benefit of retifanlimab for the treatment of patients with advanced or metastatic SCAC.

In addition to SCAC, retifanlimab is also currently under evaluation as a monotherapy for patients with microsatellite instability-high endometrial cancer, and Merkel cell carcinoma; and in combination with platinum-based chemotherapy for patients with non-small cell lung cancer. Incyte has an exclusive collaboration and license agreement with MacroGenics, Inc. for global rights to retifanlimab and a collaboration and license agreement with Zai Lab for the development and commercialization of retifanlimab in Greater China.

Need help keeping up to date on US and EU approvals?

The Antibody Society maintains a comprehensive table of approved monoclonal antibody therapeutics and those in regulatory review in the EU or US in the Web Resources section of our website.

US approval for mogamulizumab

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On August 8, 2018, the U.S. Food and Drug Administration (FDA) approved Poteligeo (mogamulizumab-kpkc) for intravenous use for the treatment of adult patients with relapsed or refractory mycosis fungoides (MF) or Sézary syndrome (SS) after at least one prior systemic therapy. The diseases are subtypes of cutaneous T-cell lymphoma (CTCL), which is a rare and difficult-to-treat type of non-Hodgkin lymphoma. The FDA had previously granted mogamulizumab Breakthrough Therapy and Orphan Drug designations, and the biologics license application for mogamulizumab received a priority review.

FDA’s approval was based on an open-label, multi-center, randomized Phase 3 clinical trial (NCT01728805) of 372 patients with relapsed MF or SS who received either mogamulizumab or vorinostat. Study sites were located in the US, Europe, Japan and Australia. Median progression-free survival was 7.6 months for patients administered mogamulizumab compared to 3.1 month for patients taking vorinostat in this clinical trial.

Developed by Kyowa Kirin, mogamulizumab is a humanized glyco-engineered monoclonal antibody that binds to CC chemokine receptor type 4 on cancer cells. Mogamulizumab was produced using Kyowa Hakko Kirin’s proprietary POTELLIGENT® platform, which produces antibodies with low / no fucose content. Such antibodies have increased affinity to FcγRIIIa (CD16), and enhanced antibody-dependent cell-mediated cytotoxicity activity. Mogamulizumab’s first approval, in 2012, was granted by the Japanese Ministry of Health, Labour and Welfare for treatment of patients with relapsed or refractory CCR4-positive adult T-cell leukemia-lymphoma.

The Antibody Society maintains a comprehensive table of approved monoclonal antibody therapeutics and those in regulatory review in the EU or US. As of Aug 8, a total of 5 antibody therapeutics had been granted first approvals in either the US or EU in 2018, and marketing applications for another 11 that have not yet been approved in either the EU or US are undergoing review in these regions. Please log in to access the table in either PDF or Excel formats, located in the Members Only section.

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