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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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Antibody immune checkpoint modulators in the clinic

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The treatment of cancer via antibody therapeutics that modulate immune responses is the focus of substantial research and development by the biopharmaceutical industry. To date, 6 monoclonal antibodies (mAbs) that function by modulating immune checkpoints have been approved in the US: ipilimumab (anti-cytotoxic T-lymphocyte-associated antigen 4 (CTLA4)); pembrolizumab and nivolumab (anti-programmed death receptor 1 (PD-1)); durvalumab, avelumab, and atezolizumab (anti-programmed death ligand 1 (PD-L1)). Cemiplimab, another anti-PD-1 mAb, is currently undergoing regulatory review. Antibody immune checkpoint modulators can be used to treat many types of cancer,[1] which makes them highly attractive for biopharmaceutical development. For example, the approved products, which target only 3 of the many proteins involved in either stimulating or inhibiting immune responses, are used to treat melanoma, non-small-cell lung cancer, head and neck cancer, Hodgkin’s lymphoma, bladder cancer, gastric/gastroesophageal junction adenocarcinoma, renal cell cancer, hepatocellular cancer, Merkel cell carcinoma and colorectal cancer. [2]

More than 80 antibody immune checkpoint modulators sponsored by commercial firms are in clinical development, and they comprise ~ 24% of the clinical pipeline of antibody therapeutics for cancer. Most are in early development, with 50 and 28 antibody immune checkpoint modulators undergoing evaluation in Phase 1 and Phase 2 clinical studies, respectively. Seven (IBI308, BCD-100, PDR001, tislelizumab, camrelizumab, utomilumab, and tremelimumab) are undergoing evaluation in late-stage studies.[3]

Despite the fact that 5 antibodies targeting the PD-1 pathway are already marketed, PD-1 and PD-L1 remain popular as targets for antibodies in development. Of the antibody immune checkpoint modulators currently in the clinic, 21 molecules target PD-1, including five in late-stage clinical studies, and 9 antibodies target PD-L1. Other popular antigens for antibodies in clinical development include glucocorticoid-induced tumor necrosis factor receptor (GITR; target of 7 antibodies); CD40, LAG-3 and OX40 (each the target of 6 antibodies); as well as T-cell immunoglobulin and mucin-domain-containing molecule (TIM-3), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) and CTLA4 (each the target of 4 antibodies). In addition, two bispecific antibodies (anti-PD-1, LAG-3 MGD013; anti-PD-L1, CTLA-4 AK104) targeting these immune checkpoints are in clinical studies; to avoid double counting, these two were excluded from the totals given above.

Over 100 antibody immune checkpoint modulators have entered commercially sponsored clinical studies since 2000, but ~60% of the molecules first entered such studies in the past 3 years. The ultimate fates (approval or termination) for most of the molecules are thus not yet known, but the available data is sufficient to calculate a Phase 1 to 2 transition rate, which is 74%. This rate compares favorably with that for all antibody therapeutics (75%) and anti-cancer antibody therapeutics (69%). The current data suggest that antibody immune checkpoint modulators, as a group, has a notably higher Phase 2 to 3 transition rate compared with all antibody therapeutics. This result, however, is based on outcomes for relatively few molecules. It should be noted that clinical studies may be terminated for business reasons, as well as safety or efficacy issues. For example, although PD-1 and PD-L1 are well-validated targets, the market for anti-PD-1 and anti-PD-L1 antibodies in the future may not be sufficient to justify continued development of all such antibodies in the current pipeline. Termination of molecules at Phase 2 for business reasons would decrease the Phase 2 to 3 transition rate. To date, no antibody immune checkpoint modulators have been terminated during regulatory review; the transition rate at that phase is thus 100%.

The Antibody Society has partnered with Hanson Wade to track trends in the clinical development of innovative cancer therapies, with a focus on immune checkpoint modulators and antibody-drug conjugates. As the date for ICI Boston 2018 (March 19-21) approaches, Hanson Wade has prepared a comprehensive e-book that provides insights into combination strategies involving immune checkpoint inhibitors, which can be downloaded here. Members of The Antibody Society qualify for a 20% discount to ICI Boston 2018. Please contact us at membership@antibodysociety.org for the code.

  1. Torphy RJ, Schulick RD, Zhu Y. Newly Emerging Immune Checkpoints: Promises for Future Cancer Therapy. Int J Mol Sci. 2017; 18(12). pii: E2642. doi: 10.3390/ijms18122642.
  2. Iwai Y, Hamanishi J, Chamoto K, Honjo T. Cancer immunotherapies targeting the PD-1 signaling pathway. J Biomed Sci 2017; 24:26. doi.org/10.1186/s12929-017-0329-9.
  3. Kaplon H, Reichert JM. Antibodies to watch in 2018. MAbs. 2018 Jan 4:1-21. doi: 10.1080/19420862.2018.1415671.

The Antibody Society tracks the progress of commercially sponsored antibody therapeutics in clinical development on a continuous basis. We collect information, including molecular composition (e.g., format, isotype, target), phase of development and indications studied, from publicly available sources (e.g., press releases, company websites, meeting abstracts, published literature, clinicaltrials.gov, regulatory agency websites). Our data are cross-checked against databases generously provided by our corporate partners, including Hanson Wade’s Beacon Targeted Therapies and the Therapeutic Antibody Database. It should be noted that companies may not publicly disclose all information for all molecules in the pipeline, especially those in the early stages of development. The numbers of molecules discussed above should thus be considered minimums, as targets have not been disclosed for all the molecules we are tracking. We look forward to reporting additional trends and metrics for antibody therapeutics development in the future.

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IMMUNO-ONCOLOGY: CHECKPOINTS

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The Antibody Society invites you to attend its annual meeting, Antibody Engineering & Therapeutics, on December 11-15, 2017 at the Manchester Grand Hyatt, San Diego, CA! In this summary, chairperson James Larrick, M.D., Ph.D., Managing Director and Chief Medical Officer, Panorama Research Institute and Velocity Pharmaceutical Development, discusses what you will learn at his session on immune-oncology checkpoints, which will be held on Friday December 15.

The management of cancer has dramatically changed over the past decade with the introduction of novel immunotherapies, chief among them inhibitors of checkpoint receptors — molecules whose function is to restrain the host immune response.  Antibodies inhibiting CTLA4 and PD1-PD-L1 have shown remarkable clinical benefit.  The field is evolving rapidly, with many clinical trials testing novel checkpoint inhibitors (e.g., anti-LAG3, anti-TIM3), alone, in combination, or with other targeted therapies. A sampling of novel approaches will be covered in this symposium.

This Friday morning (December 15, 2017) session will be led off by Mickey Hu (Panorama Institute of Molecular Medicine) who has developed a series of novel immunomodulatory drugs that suppress PD-L1 expression in tumor cells and inhibit the PD-L1/PD-1 checkpoint, resulting in the recruitment of natural killer (NK) cells into the tumor microenvironment that leads to tumor suppression. Efforts to combine immunomodulatory drugs with checkpoint blockades to overcome difficult-to-treat cancers with tolerable side effects will be described.

Clinical lead candidate antibodies often lack species cross-reactivity, necessitating the development of substitute antibodies for pre-clinical development in mice or monkeys. Next, Erik Hofman, (Argenx) will describe the use of the SIMPLE Antibody platform to generate functional human-mouse cross-reactive antibodies against several validated immune checkpoint proteins, including PD-1, VISTA and LAG-3.

Xin Lu (University of Notre Dame) will present data indicating that targeted therapy against myeloid-derived suppressor cells, using multikinase inhibitors such as cabozantinib and dactolisib, can synergize with immune checkpoint blockade antibodies (e.g., anti-CTLA4, anti-PD1) to eradicate metastatic castration-resistant prostate cancer.

A key feature of effective cancer immunotherapy relies on enhanced anti-tumor immune response and reduced suppressive effects. As natural cytokines are made to maintain a balance between activation and suppression, they are often unable to achieve desired therapeutic efficacy. Cheng-I Wang (Biomedical Sciences Institutes, ASTAR, Singapore) will describe a cytokine receptor agonist antibody that mimics IL-2’s immune stimulatory effects on CD8 T cells with minimal Treg activation.

Cow antibodies have unusually long CDR3 regions. Vaughn Smider (The Scripps Research Institute) has characterized the genetic and structural properties of these antibodies, and has identified novel antibodies against HIV and exhausted T-cell targets utilizing this approach.

The final speaker, Sarah Crome, (Princess Margaret Cancer Centre, University Health Network, Canada) will describe efforts to characterize a unique innate lymphoid cell (ILC) population that suppresses the expansion and function of tumor-associated T cells, and is associated with early recurrence in high-grade cancer. This regulatory ILC population has properties that overlap with NK cells and other defined ILCs, yet can be differentiated by a distinct gene expression signature. Studies defining molecular interactions that control regulatory ILC function and ways to target this population to enhance immunotherapy will be presented.

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Membership is free for students and employees of the Society’s corporate sponsors.