ZAP70

Preclinical data highlight potential target engagement biomarkers to support clinical evaluation of CBL-B inhibitors BOSTON, Nov. 3, 2023 /PRNewswire/ -- HotSpot Therapeutics, Inc., a biotechnology company pioneering the discovery and development of oral, small molecule allosteric therapies targeting regulatory sites on proteins referred to as "natural hotspots," today announced the presentation of additional preclinical data from the Company's Casitas B-lineage lymphoma proto-oncogene (CBL-B) program in a poster presentation at the 2023 Society for Immunotherapy of Cancer (SITC) Annual Meeting. "Our enthusiasm for the therapeutic potential of HST-1011, a potential best-in-class CBL-B inhibitor, is partly driven by our belief that it can drive single-agent biological activity," said Timothy Reilly, Ph.D., Chief Development Officer of HotSpot. "Understanding which peripheral biomarkers are associated with this biology and the reliability of their measurement is critical to our characterization of of HST-1011's activity in patients as we advance our ongoing Phase 1/2 study." The presentation describes preclinical data in which HotSpot CBL-B inhibitors were evaluated in both in vitro and in vivo settings to identify and then characterize a repertoire of potential proximal biomarkers of CBL-B inhibitory activity. Several proteins, including phosphorylated ZAP70, Notch1 and IGF1R, showed robust dose-dependent effects and were further assessed for their reliability and potential feasibility for use in a clinical setting. The results provide support for their potential use in evaluating HST-1011 in our ongoing Phase 1/2, open-label, clinical study designed to evaluate HST-1011 alone and subsequently in combination with Regeneron's anti-PD-1 therapy, Libtayo® (cemiplimab), in patients with advanced solid tumors that are relapsed on or are refractory to anti-PD(L)-1 or standard of care therapies. Information on this clinical trial can be found on (NCT05662397). About HST-1011 HST-1011 is an investigational orally bioavailable, selective, small molecule allosteric inhibitor of CBL-B, an E3 ubiquitin protein ligase critically involved in immune cell response. Because CBL-B functions as a master regulator of effector cell (T cell and natural killer cell) immunity, its inactivation removes its endogenous negative regulatory functions to substantially enhance anti-tumor immunity. Preclinical data has demonstrated HST-1011's ability to bind to and inhibit a natural hotspot on CBL-B, yielding the activation and propagation of a targeted anti-tumor immune response. Enabled by HotSpot's proprietary Smart Allostery™ platform, HST-1011 is designed with tight binding, low nanomolar potency, a slow dissociation rate from the target to enable sustained pharmacology, and greater selectivity for CBL-B relative to C-CBL. About HotSpot Therapeutics, Inc. HotSpot Therapeutics, Inc. is pioneering a new class of allosteric drugs that target certain naturally occurring pockets on proteins called "natural hotspots." These pockets are decisive in controlling a protein's cellular function and have significant potential for new drug discovery by enabling the systematic design of potent and selective small molecules with novel pharmacology. The Company's proprietary Smart Allostery™ platform combines computational approaches and AI-driven data mining of large and diverse data sets to uncover hotspots with tailored pharmacology toolkits and bespoke chemistry to drive the rapid discovery of novel hotspot-targeted small molecules. Leveraging this approach, HotSpot is building a broad pipeline of novel allosteric therapies for the treatment of cancer and autoimmune diseases. To learn more, visit . Investor & Media Contact: Natalie Wildenradt [email protected] SOURCE HotSpot Therapeutics

- Preclinical data identify SHP-1 as a downstream effector of GDF-15-mediated inhibition of T cell adhesion, further elucidating mechanistic details of GDF-15’s role in immunotherapy resistance. - Combination of visugromab and bispecific T-cell engager significantly increased the number of intratumoral T cells in a mouse tumor model, providing a strong rationale to further investigate this combination approach. - Preclinical data continue to expand the potential of GDF-15 neutralizing therapy, supporting its role as a critical component for treatment success in a broad range of anti-cancer regimens. MUNICH--(BUSINESS WIRE)-- CatalYm today announced the presentation of two preclinical data sets expanding the mechanistic understanding and clinical application of its lead anti-GDF-15 antibody candidate, visugromab, at the upcoming Society for Immunotherapy of Cancer (SITC) 2023 Annual Meeting in San Diego, USA. The findings will be presented in two poster sessions on Friday, November 3rd, and Saturday, November 4th, and provide further clarification on GDF-15-mediated inhibition of T cell infiltration and highlight the potential of combining visugromab with bispecific T-cell engagers as a novel approach in cancer therapy. Visugromab is currently evaluated in a broad Phase 2 program in combination with anti-PD-1 treatment in multiple solid tumor indications. “We are advancing our understanding of the underlying molecular mechanisms that are the foundation for GDF-15’s central role in tumor resistance to immunotherapy and visugromab’s potential to reinstate anti-tumor activity and enhance responses,” said Dr. Christine Schuberth-Wagner, Chief Scientific Officer at CatalYm. “The new data for the combination of visugromab with bispecific T-cell engagers provide strong scientific support for a synergistic effect in this novel therapeutic setting. We will further investigate this combination, which could extend durability and improve outcomes for a broad range of cancer patients with high medical need.” Poster Presentation Identifying SHP-1 as Central Mediator of GDF-15-related T Cell Adhesion Inhibition The first data set further expands understanding of visugromab’s mechanism of action and the underlying pathways involved in the development of GDF-15-mediated therapy resistance in cancer cells. When investigating the involvement of additional cell adhesion pathway components downstream of GDF-15, SHP-1 was identified as a central mediator of GDF-15-related inhibition of T cell adhesion. SHP-1 is an intracellular tyrosine phosphatase known as a negative regulator of antigen-dependent activation and proliferation of T cells. Inhibition of SHP-1 resulted in abrogation of GDF-15’s inhibitory effect. The researchers confirmed that GDF-15 via SHP-1 inhibits the phosphorylation of ZAP-70, another intracellular tyrosine kinase involved in LFA-1 inside-out activation and T cell receptor signaling. These data connect SHP-1 as a downstream effector to the recently published GDF-15-mediated reduction of high-affinity LFA-1 conformation, leading to impaired endothelial adhesion and transmigration of T cells toward the tumor site. Poster Presentation Details Title: SHP-1 is a central mediator of GDF-15 mediated adhesion inhibition in T cells Presenter: Dr. Christine Schuberth-Wagner Abstract number: 1049 Date and time: Friday, Nov 3rd, 5:00-6:30pm PDT Poster Presentation on Visugromab/T-Cell Engager Combination The second data set supports the expansion of visugromab’s application to combinations with immune cell-engaging therapies, underlining its potential to be a critical component for treatment success in a broad range of anti-cancer regimens. The preclinical analyses investigated a new combination of visugromab with a bispecific T-cell engager (tebentafusp). Anti-tumor activity of bispecific T-cell engagers is dependent on infiltration of effector T cells into the tumor microenvironment. In vivo analyses in a mouse tumor model demonstrated that the combination with visugromab significantly increased the number of intratumoral T cells compared with tebentafusp alone. This indicates a direct positive impact of visugromab’s infiltration-enhancing activity on the anti-tumor activity of bispecific T-cell engaging treatments and provides a strong rationale to further investigate this therapeutic concept. Poster Presentation Details Title: GDF-15 neutralizing antibody visugromab increases intratumoral immune cell infiltration to support bispecific T-cell engagers Presenter: Dr. Christine Schuberth-Wagner Abstract number: 1196 Date and time: Saturday, Nov 4th, 7:00-8:30pm PDT CatalYm is investigating its GDF-15-neutralizing antibody visugromab in a broad Phase 2 clinical program, the GDFather (GDF-15 Antibody-mediaTed Human Effector Cell Relocation) trials, in combination with the anti-PD-1 inhibitor nivolumab in patients with advanced solid tumors. First interim data from the Phase 2 (NCT04725474) trial continue to demonstrate a very good safety and tolerability pro promising early responses in major cancer indications, including non-small cell lung cancer (NSCLC), bladder cancer and hepatocellular carcinoma (HCC). In addition, CatalYm recently announced an exploratory Phase 2 study, GDFather-NEO (NCT06059547), evaluating visugromab in combination with neoadjuvant immunotherapy in first-line muscle-invasive bladder cancer. About Visugromab (CTL-002) Visugromab is a monoclonal antibody that neutralizes the tumor-derived Growth Differentiation Factor-15 (GDF-15), a locally acting immunosuppressant fostering immunotherapy resistance. Neutralizing GDF-15 with visugromab reverses key cancer resistance mechanisms to reinstate an efficient anti-tumor response by reenabling immune cell activation and tumor infiltration. Visugromab has already demonstrated a good safety pro potent and durable anti-tumor efficacy in combination with anti-PD-1 treatment in advanced cancer patients The antibody is currently being investigated in ongoing Phase 2 studies in multiple solid tumor indications. About CatalYm CatalYm has identified GDF-15 as a key cancer therapy resistance mechanism and is developing a safe and efficacious immune therapy for solid tumors. GDF-15, an immunosuppressant important for feto-maternal tolerance, is hijacked by cancer cells to evade immune system attack. Visugromab, CatalYm’s lead antibody, has demonstrated durable anti-tumor efficacy with long-lasting objective responses in relapsed and refractory metastatic solid tumor patients in combination with anti-PD-1. CatalYm is now advancing to Phase 2b studies to confirm visugromab as a new class of cancer immunotherapy in a broad range of anti-cancer regimens.

According to new research, T cells have a nuclear receptor doing something very odd—but very important—to help them fight pathogens and destroy cancer cells. This receptor, called retinoic acid receptor alpha, is known to control gene expression programs in the nucleus, but it also now appears to operate outside the cell nucleus to coordinate the early events triggered at the cell surface that lead to T cell activation. According to new research in the journal Immunity, T cells have a nuclear receptor doing something very odd -- but very important -- to help them fight pathogens and destroy cancer cells. This receptor, called retinoic acid receptor alpha (RARα), is known to control gene expression programs in the nucleus, but it also now appears to operate outside the cell nucleus to coordinate the early events triggered at the cell surface that lead to T cell activation. A T cell (pictured) can have many roles in the immune system. (Image courtesy NIAID) Scientists wouldn't normally expect to see a nuclear receptor such as RARα playing this role outside the cell nucleus. And yet the new findings suggest T cells cannot begin to fight disease without a form of RARα on the scene in the cytoplasm. "Cytoplasmic retinoic acid receptors turn out to be central for a T cell to link sensing at the cell surface with downstream signaling cascades and gene expression programs that transform the T cell to become an active fighter," says Professor Hilde Cheroutre, Ph.D., who led the new study at La Jolla Institute for Immunology (LJI) with LJI Assistant Professor Samuel Myers, Ph.D., LJI Professor Mitchell Kronenberg, Ph.D., and LJI Professor Emeritus Amnon Altman, Ph.D. The study is also the result of a successful collaboration with scientists at the RIKEN Center for Integrative Medical Sciences in Japan and local teams at UC San Diego and the Salk Institute. Helping T cells respond to danger To understand this finding, it helps to picture the geography of a T cell. The cell nucleus (with its bundled-up DNA) sits in the middle of the cell. Other molecules and cellular structures called organelles float in the cytoplasm outside the nucleus surrounded by a membrane at the border of the cell (cell membrane). Special molecules called T cell receptors (TCRs) sit on the cell membrane, where they receive messages from other cells. You can imagine TCRs as fire-spotters, the lookouts who scan for smoke from remote cabins in the wilderness. Just as fire-spotters need to alert officials to any smoke in the distance, TCRs need to quickly signal headquarters -- the cell nucleus -- if they detect a potential threat, such as a virus or cancer cell. Sending that signal to the cell nucleus is critical for activating gene expression to transform the T cell to a fighter cell. But TCRs can't just pick up a phone, so how do they alert the distant cell nucleus to trouble? The signaling process is fascinating. Once the TCR is triggered, molecules called kinases (enzymes that add phosphates to proteins) work with adaptors that tell nearby proteins to "click" together and assemble a special molecular "activation complex." This complex is called a TCR signalosome, and it comes together just inside the cell membrane. "The TCR signalosome is extremely important for mediating communication between the outside and the inside of a cell," says Cheroutre. And although the TCR signalosome has been studied by many people for many, many years, no one had ever detected RARα in this activation complex before. "This new finding will change the way we think about TCR signals," says Kronenberg. Uncovering RARα's role RARα's secret came to light thanks to the development of CRISPR techniques, advances in imaging and mass spectrometry -- and years of hard work from the LJI team and collaborators. RARα belongs to a large family of retinoic acid receptors that normally sit on control regions of target genes in the nucleus. These retinoic acid receptors recruit repressor and activator molecules that let them "switch off" or "switch on" expression of these target genes. This important job in the nucleus has earned retinoic acid receptors the title of "nuclear receptors." Several years ago, Cheroutre and her colleagues published research showing that retinoic acid (RA), which we get from vitamin A, triggers nuclear RARα and gene expression important for the differentiation of suppressive regulatory T cells that reduce the immune response. For the new study, Cheroutre wanted to investigate how retinoic acid controls that suppressive T cell fate, so her laboratory started to look at retinoic acid receptors, such as RARα, more closely. The researchers were intrigued to find that RARα actually comes in two variants, called isoforms. "These isoforms are encoded by the same gene, but they differ a little bit at one end. The consequences however are more significant and lock one form in the cytoplasm whereas the other form is confined to the nucleus" says Cheroutre. Could these two isoforms play different roles in T cells? Looking closer, the researchers realized this RARα isoform did not respond to retinoic acid and didn't even have the right equipment to function as a nuclear receptor. "It didn't have the tools that are important for nuclear receptors, namely the ability to interact with DNA and the ability to translocate from the cytoplasm to the nucleus," says Cheroutre. Using CRISPR gene editing techniques to modulate expression of the two isoforms, the researchers found that modulating the cytoplasmic isoform caused major problems for TCR signaling in the cytoplasm and impaired the communication with the control center in the nucleus. RARα is in the right place at the right time Once they knew where to look for this RARα isoform, the researchers tried to figure out what other proteins RARα interacted with. This work revealed interactions with the kinase ZAP70, a major component of the TCR signalosome. Another important, early clue that RARα had a role outside of the nucleus came from proteomics work led by the Myers Lab. The researchers used techniques in mass spectrometry to detect a process called phosphorylation, which is when a molecule called a phosphoryl group is attached to proteins by an upstream kinase. For T cells, phosphorylation spurs key proteins into action when a threat is near. "There are hundreds to thousands of dynamic phosphorylation events that occur during the first hour or so of T cell stimulation," says Myers. As Myers and his colleagues examined their data, they were surprised to spot a phosphorylation event related to RARα. In fact, phosphorylation of RARα began just three minutes into T cell activation. "Because this event was that early, our findings suggest that this phosphorylation of RARα is near the T cell receptor -- and it has a burst in activity right after the TCR is stimulated," says Myers. This discovery added to the evidence that the cytoplasmic isoform of RARα is activated by the TCR instead of by RA like the nuclear RARα. Thus, this new form of RARα represents an essential component of the TCR/ZAP70 activation complex at the cell surface. Cheroutre and her colleagues then shed light on another fascinating phenomenon in the cytoplasm. Scientists knew that RA, which is present in the blood and taken up by the T cells, is further transported to the nucleus by a molecule called cellular retinoic acid binding protein 2 (CRABP2). CRABP2 in the cytoplasm binds to RA and carries it into the cell nucleus where it activates nuclear RARα. The researchers showed that without CRABP2, RA remains in the cytoplasm of the T cell and instead of activating cytoplasmic RARα, it interferes with TCR-activated RARα in the cytoplasm and blocks T cell activation. As a result, the T cell can no longer effectively fight infections or kill cancer cells. The good side of this phenomenon is that the interference of RA with the cytoplasmic RARα reduces the inflammatory response of the T cell. The researchers think this process may make an important target to fight autoimmune diseases and other inflammatory diseases. Next steps for understanding TCR signaling The new study emphasizes the importance of understanding not just a receptor's location but its special talents and interacting partners. "Retinoic acid receptors are really 'complex formers' and it all depends on who they recruit or reject. They have the unique capacity to engage molecules and release other molecules," says Cheroutre. "Retinoic acid receptors are the 'architects' of complexes." The current study focused on T cells, but it may have implications for understanding signaling in many other cell types. For example, the Cheroutre Lab has gone on to show over-expression of this RARα isoform in the cytoplasm of cancer cells. What is extranuclear RARα doing there? What signals does it help relay? "Understanding the role of this receptor has such major implications for studying protective immunity, anticancer therapies, autoimmune disease, and neurological diseases," says Cheroutre. The Myers Lab will spearhead the next steps in this research. Myers hopes to figure out exactly what RARα does in the TCR/ZAP70 signaling complex. "Does it modulate the signaling strength or duration? How does that change in signal propagate to gene expression and T cell activity?" he asks. His laboratory plans to use a "systems biochemistry" approach to further study the role of cytoplasmic and nuclear RARα in T cell activation and differentiation. "The ultimate goal is to see if we can identify a new pathway, or set of pathways, that could be exploited to control autoimmune diseases and inflammation or enhance protective immunity to eradicate tumors or fight infections," says Myers. Additional authors of the study, "A regulatory circuit controlled by extranuclear and nuclear retinoic acid receptor α determines T cell activation and function," are co-first authors Alexandre Larange, Ikuo Takazawa, Kiyokazu Kakugawa, and Nicolas Thiault; and co-authors Soomun Ngoi, Meagan E. Olive, Hitoshi Iwaya, Laetitia Seguin, Ildefonso Vicente-Suarez, Stephane Becart, Greet Verstichel, Ann Balancio, Amnon Altman, John T. Chang, Ichiro Taniuchi, and Bjorn Lillemeier. This study was supported by the U.S. National Institutes of Health (grants U01 AI125957, P01 DK46763, and R01 AI106298), the Philippe Foundation, and a CCFA Fellowship (697275).