Journal Articles

Coupling IL-2 with IL-10 to mitigate toxicity and enhance antitumor immunity Spotlight 

Ahn et al. showed in vitro (using human PBMCs) and in mice that IL-10 suppressed IL-2 induction of CRS-associated cytokines by suppressing TNFα production while potentiating IL-2-mediated antitumor activities. DK210(EGFR) – a fusion protein comprising IL-2 coupled to a high-affinity IL-10 mutein targeted by an anti-EGFR scFv scaffold to tumor cells – activated CTLs and NK cells, increased perforin/granzyme B secretion, limited Treg expansion, boosted the CD8+ T cell/Treg ratio within tumors, sustained CTL functions, and enhanced efficacy in murine tumor models. In NHP, at projected therapeutic doses, DK210(EGFR) induced immune activation without inducing CRS or significant organ toxicity.

Contributed by Paula Hochman

Ahn et al. showed in vitro (using human PBMCs) and in mice that IL-10 suppressed IL-2 induction of CRS-associated cytokines by suppressing TNFα production while potentiating IL-2-mediated antitumor activities. DK210(EGFR) – a fusion protein comprising IL-2 coupled to a high-affinity IL-10 mutein targeted by an anti-EGFR scFv scaffold to tumor cells – activated CTLs and NK cells, increased perforin/granzyme B secretion, limited Treg expansion, boosted the CD8+ T cell/Treg ratio within tumors, sustained CTL functions, and enhanced efficacy in murine tumor models. In NHP, at projected therapeutic doses, DK210(EGFR) induced immune activation without inducing CRS or significant organ toxicity.

Contributed by Paula Hochman

ABSTRACT: Wild-type interleukin (IL)-2 induces anti-tumor immunity and toxicity, predominated by vascular leak syndrome (VLS) leading to edema, hypotension, organ toxicity, and regulatory T cell (Treg) expansion. Efforts to uncouple IL-2 toxicity from its potency have failed in the clinic. We hypothesize that IL-2 toxicity is driven by cytokine release syndrome (CRS) followed by VLS and that coupling IL-2 with IL-10 will ameliorate toxicity. Our data, generated using human primary cells, mouse models, and non-human primates, suggest that coupling of these cytokines prevents toxicity while retaining cytotoxic T cell activation and limiting Treg expansion. In syngeneic murine tumor models, DK210 epidermal growth factor receptor (EGFR), an IL-2/IL-10 fusion molecule targeted to EGFR via an anti-EGFR single-chain variable fragment (scFV), potently activates T cells and natural killer (NK) cells and elicits interferon (IFN)γ-dependent anti-tumor function without peripheral inflammatory toxicity or Treg accumulation. Therefore, combining IL-2 with IL-10 uncouples toxicity from immune activation, leading to a balanced and pleiotropic anti-tumor immune response.

Author Info: (1) Deka Biosciences, Inc., Germantown, MD, USA. (2) Deka Biosciences, Inc., Germantown, MD, USA. (3) Deka Biosciences, Inc., Germantown, MD, USA. (4) Deka Biosciences, Inc., Germa

Author Info: (1) Deka Biosciences, Inc., Germantown, MD, USA. (2) Deka Biosciences, Inc., Germantown, MD, USA. (3) Deka Biosciences, Inc., Germantown, MD, USA. (4) Deka Biosciences, Inc., Germantown, MD, USA. (5) Deka Biosciences, Inc., Germantown, MD, USA. (6) Deka Biosciences, Inc., Germantown, MD, USA. (7) Deka Biosciences, Inc., Germantown, MD, USA. (8) Deka Biosciences, Inc., Germantown, MD, USA. (9) Deka Biosciences, Inc., Germantown, MD, USA. (10) Deka Biosciences, Inc., Germantown, MD, USA. (11) Deka Biosciences, Inc., Germantown, MD, USA. (12) Deka Biosciences, Inc., Germantown, MD, USA. (13) Deka Biosciences, Inc., Germantown, MD, USA. (14) Deka Biosciences, Inc., Germantown, MD, USA. (15) Deka Biosciences, Inc., Germantown, MD, USA. (16) Deka Biosciences, Inc., Germantown, MD, USA. (17) Deka Biosciences, Inc., Germantown, MD, USA. (18) Deka Biosciences, Inc., Germantown, MD, USA. (19) Deka Biosciences, Inc., Germantown, MD, USA. (20) Deka Biosciences, Inc., Germantown, MD, USA. (21) Deka Biosciences, Inc., Germantown, MD, USA. (22) Deka Biosciences, Inc., Germantown, MD, USA. (23) Deka Biosciences, Inc., Germantown, MD, USA. (24) Deka Biosciences, Inc., Germantown, MD, USA. Electronic address: mummj@dekabiosciences.com.

Bispecific killer cell engager-secreting CAR-T cells redirect natural killer specificity to enhance antitumour responses Spotlight 

Testing various combinations, Fan et al. found that the administration of bispecific killer cell-engager (BiKE)-secreting CAR T cells alongside weekly injections of NK cells was optimal for achieving long-term control in murine hematologic tumor models. In a solid tumor model, NK cells co-administred with BiKE+ CAR T cells showed tumor parenchyma infiltration, whereas NK cells co-administred with BiKE- CAR T cells were primarily found in the peritumoral connective tissue. The simultaneous expression of a CD19-targeting CAR and EGFR-targeting BIKEs in T cell led to complete eradication of heterogeneous EGFR+CD19- and EGFR-CD19+ tumor cells in vivo.

Contributed by Ute Burkhardt

Testing various combinations, Fan et al. found that the administration of bispecific killer cell-engager (BiKE)-secreting CAR T cells alongside weekly injections of NK cells was optimal for achieving long-term control in murine hematologic tumor models. In a solid tumor model, NK cells co-administred with BiKE+ CAR T cells showed tumor parenchyma infiltration, whereas NK cells co-administred with BiKE- CAR T cells were primarily found in the peritumoral connective tissue. The simultaneous expression of a CD19-targeting CAR and EGFR-targeting BIKEs in T cell led to complete eradication of heterogeneous EGFR+CD19- and EGFR-CD19+ tumor cells in vivo.

Contributed by Ute Burkhardt

ABSTRACT: T cells and natural killer (NK) cells collaborate to maintain immune homeostasis. Current cancer immunotherapies predominantly rely on the individual application of these cells. Here we use bicistronic vectors to co-express chimeric antigen receptors (CARs) and secreted immune cell engagers (ICEs), leveraging the combined therapeutic potential of both effector cell types. After in vitro validation of immune cell engager secretion and function, various combinatorial approaches are systematically compared in mouse models, identifying a highly effective combination of bispecific killer cell engager (BiKE)-secreting CAR-T cells and NK cells. Beyond a simple combination of conventional CAR-T cells and NK cells, this strategy demonstrates superior efficacy in CD19(+) B cell leukaemia and lymphoma and EGFR(+) solid tumour models while reducing the dosage dependence on CAR-T cells. Moreover, CAR-T cells and BiKEs targeting distinct antigens exhibit suppression of tumour cells with heterogeneous antigen expression. These findings indicate that combining BiKE-secreting CAR-T cells and NK cells offers a promising strategy to combat tumour antigen heterogeneity and immune evasion.

Author Info: (1) Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. Liangzhu Laboratory, Zhejiang University, Hangzhou,

Author Info: (1) Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. Liangzhu Laboratory, Zhejiang University, Hangzhou, China. (2) Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. Liangzhu Laboratory, Zhejiang University, Hangzhou, China. Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, China. Institute of Hematology, Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Zhejiang University, Hangzhou, China. (3) Liangzhu Laboratory, Zhejiang University, Hangzhou, China. Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, China. Institute of Hematology, Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Zhejiang University, Hangzhou, China. (4) Liangzhu Laboratory, Zhejiang University, Hangzhou, China. Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, China. Institute of Hematology, Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Zhejiang University, Hangzhou, China. (5) Liangzhu Laboratory, Zhejiang University, Hangzhou, China. Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, China. Institute of Hematology, Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Zhejiang University, Hangzhou, China. (6) Liangzhu Laboratory, Zhejiang University, Hangzhou, China. Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, China. Institute of Hematology, Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Zhejiang University, Hangzhou, China. (7) School of Public Health, Zhejiang University School of Medicine, Hangzhou, China. (8) School of Public Health, Zhejiang University School of Medicine, Hangzhou, China. (9) Center for Cell Engineering and Immunology Program, Sloan Kettering Institute, New York, NY, USA. (10) Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. huanghe@zju.edu.cn. Liangzhu Laboratory, Zhejiang University, Hangzhou, China. huanghe@zju.edu.cn. Institute of Hematology, Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Zhejiang University, Hangzhou, China. huanghe@zju.edu.cn. (11) Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. sunj4@zju.edu.cn. Liangzhu Laboratory, Zhejiang University, Hangzhou, China. sunj4@zju.edu.cn. Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, China. sunj4@zju.edu.cn. Institute of Hematology, Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Zhejiang University, Hangzhou, China. sunj4@zju.edu.cn.

Sensitization of tumours to immunotherapy by boosting early type-I interferon responses enables epitope spreading Spotlight 

Qdaisat, Wummer, and Stover et al. demonstrated that early type-I interferon responses restored a defective damage response in poorly immunogenic tumors to enable epitope spreading and sensitivity to ICB. ICB response was transferrable to resistant models due to epitope spread against poorly immunogenic tumor antigens in an IFNAR1-dependent manner. Systemic administration of lipid particles loaded with RNA coding for tumor-unspecific antigens enhanced interferon responses, induced epitope spreading, and reprogrammed the TME from myeloid suppressive to pro-inflammatory, favoring effector T cells for sustained immune response in poorly immunogenic models.

Contributed by Shishir Pant

Qdaisat, Wummer, and Stover et al. demonstrated that early type-I interferon responses restored a defective damage response in poorly immunogenic tumors to enable epitope spreading and sensitivity to ICB. ICB response was transferrable to resistant models due to epitope spread against poorly immunogenic tumor antigens in an IFNAR1-dependent manner. Systemic administration of lipid particles loaded with RNA coding for tumor-unspecific antigens enhanced interferon responses, induced epitope spreading, and reprogrammed the TME from myeloid suppressive to pro-inflammatory, favoring effector T cells for sustained immune response in poorly immunogenic models.

Contributed by Shishir Pant

ABSTRACT: The success of cancer immunotherapies is predicated on the targeting of highly expressed neoepitopes, which preferentially favours malignancies with high mutational burden. Here we show that early responses by type-I interferons mediate the success of immune checkpoint inhibitors as well as epitope spreading in poorly immunogenic tumours and that these interferon responses can be enhanced via systemic administration of lipid particles loaded with RNA coding for tumour-unspecific antigens. In mice, the immune responses of tumours sensitive to checkpoint inhibitors were transferable to resistant tumours and resulted in heightened immunity with antigenic spreading that protected the animals from tumour rechallenge. Our findings show that the resistance of tumours to immunotherapy is dictated by the absence of a damage response, which can be restored by boosting early type-I interferon responses to enable epitope spreading and self-amplifying responses in treatment-refractory tumours.

Author Info: (1) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. Univer

Author Info: (1) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. University of Florida Genetics Institute, University of Florida, Gainesville, FL, USA. (2) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (3) Department of Pediatrics, Division of Pediatric Hematology-Oncology, UF Health Cancer Center, University of Florida, Gainesville, FL, USA. (4) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (5) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (6) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (7) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (8) Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. (9) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (10) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (11) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (12) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (13) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (14) Department of Pediatrics, Division of Pediatric Hematology-Oncology, UF Health Cancer Center, University of Florida, Gainesville, FL, USA. (15) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (16) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (17) Department of Pediatrics, Division of Pediatric Hematology-Oncology, UF Health Cancer Center, University of Florida, Gainesville, FL, USA. (18) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (19) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (20) Department of Mechanical and Aerospace Engineering, University of Florida, Gainesville, FL, USA. (21) Department of Medicine, Division of Hematology and Oncology, UF Health Cancer Center, University of Florida, Gainesville, FL, USA. (22) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (23) Department of Pediatrics, Division of Pediatric Hematology-Oncology, UF Health Cancer Center, University of Florida, Gainesville, FL, USA. (24) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (25) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (26) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (27) Department of Pediatrics, Division of Pediatric Hematology-Oncology, UF Health Cancer Center, University of Florida, Gainesville, FL, USA. (28) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (29) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (30) College of Veterinary Medicine, University of Florida, Gainesville, FL, USA. (31) Department of Pathology, University of Florida, Gainesville, FL, USA. (32) Center of Immunotherapy and Precision Immuno-Oncology/Head and Neck Institute, Cleveland Clinic, Cleveland, OH, USA. (33) Department of Mechanical and Aerospace Engineering, University of Florida, Gainesville, FL, USA. (34) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (35) Department of Pediatrics, Division of Pediatric Hematology-Oncology, UF Health Cancer Center, University of Florida, Gainesville, FL, USA. (36) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (37) Department of Biostatistics, University of Florida, Gainesville, FL, USA. (38) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (39) Department of Pediatrics, Division of Pediatric Hematology-Oncology, UF Health Cancer Center, University of Florida, Gainesville, FL, USA. (40) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. (41) Lillian S. Wells Department of Neurosurgery, Preston A. Wells, Jr. Center for Brain Tumor Therapy, McKnight Brain Institute, University of Florida, Gainesville, FL, USA. Elias.Sayour@neurosurgery.ufl.edu. Department of Pediatrics, Division of Pediatric Hematology-Oncology, UF Health Cancer Center, University of Florida, Gainesville, FL, USA. Elias.Sayour@neurosurgery.ufl.edu.

Design of high-specificity binders for peptide-MHC-I complexes Featured  

Liu, Greenwood, Bonzanini, et al. utilized new generative AI protein design platforms to identify binders to pMHCI complexes with high specificity, overcoming many of the challenges associated with identifying high-affinity and highly-specific TCRs. When incorporated into CARs expressed on T cells, these binders could effectively stimulate T cell activation and induce target-specific cell killing, demonstrating potential for use in immunotherapies.

Liu, Greenwood, Bonzanini, et al. utilized new generative AI protein design platforms to identify binders to pMHCI complexes with high specificity, overcoming many of the challenges associated with identifying high-affinity and highly-specific TCRs. When incorporated into CARs expressed on T cells, these binders could effectively stimulate T cell activation and induce target-specific cell killing, demonstrating potential for use in immunotherapies.

ABSTRACT: Class I major histocompatibility complex (MHC-I) molecules present peptides derived from intracellular antigens on the cell surface for immune surveillance. Proteins that recognize peptide-MHC-I (pMHCI) complexes with specificity for diseased cells could have considerable therapeutic utility. Specificity requires recognition of outward-facing amino acid residues within the disease-associated peptide as well as avoidance of extensive contacts with ubiquitously expressed MHC. We used RFdiffusion to design pMHCI-binding proteins that make extensive contacts with the peptide and identified specific binders for 11 target pMHCs starting from either experimental or predicted pMHCI structures. Upon incorporation into chimeric antigen receptors, designs for eight targets conferred peptide-specific T cell activation. Our approach should have broad utility for both protein- and cell-based pMHCI targeting.

Author Info: (1) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (2) Department of Biochemistry

Author Info: (1) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (2) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (3) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. Bioengineering Graduate Program, University of Washington, Seattle, WA, USA. (4) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. Bioengineering Graduate Program, University of Washington, Seattle, WA, USA. (5) Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA. (6) Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA. (7) Departments of Molecular and Cellular Physiology and Structural Biology, Stanford University School of Medicine, Stanford, CA, USA. (8) Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, USA. Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. (9) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (10) Departments of Molecular and Cellular Physiology and Structural Biology, Stanford University School of Medicine, Stanford, CA, USA. (11) Paul G. Allen School of Computer Science and Engineering, University of Washington, Seattle, WA, USA. (12) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (13) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. (14) Paul G. Allen School of Computer Science and Engineering, University of Washington, Seattle, WA, USA. Department of Physics, University of Washington, Seattle, WA, USA. Department of Applied Mathematics, University of Washington, Seattle, WA, USA. Fred Hutchinson Cancer Center, Seattle, WA, USA. (15) Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA. Weill Cornell Medicine, New York, NY, USA. (16) Departments of Molecular and Cellular Physiology and Structural Biology, Stanford University School of Medicine, Stanford, CA, USA. Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA. (17) Department of Biochemistry, University of Washington, Seattle, WA, USA. Institute for Protein Design, University of Washington, Seattle, WA, USA. Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA.

IL-12 mRNA-LNP promotes dermal resident memory CD4+ T cell development

Spotlight 

Zabala-Peñafiel, Gonzalez-Lombana et al. showed that mice vaccinated s.c. with LNPs containing mRNA encoding leishmanial PEPCK antigen generated specific dLN and splenic Th1 and T follicular helper cells, but few dermal resident memory T cells (dTrm). These mice did not mount delayed-type hypersensitivity (DTH, which requires dTrm in non-inflamed skin) or protective responses to L. major intradermal infection. Delivering IL-12 mRNA-LNPs with PEPCK vaccines boosted levels of specific Th1 cells with skin-homing (selectin+) and memory markers in LN, increased dTrm cells in inflamed and non-inflamed skin, and induced DTH and protective responses to Leishmania challenge.

Contributed by Paula Hochman

Zabala-Peñafiel, Gonzalez-Lombana et al. showed that mice vaccinated s.c. with LNPs containing mRNA encoding leishmanial PEPCK antigen generated specific dLN and splenic Th1 and T follicular helper cells, but few dermal resident memory T cells (dTrm). These mice did not mount delayed-type hypersensitivity (DTH, which requires dTrm in non-inflamed skin) or protective responses to L. major intradermal infection. Delivering IL-12 mRNA-LNPs with PEPCK vaccines boosted levels of specific Th1 cells with skin-homing (selectin+) and memory markers in LN, increased dTrm cells in inflamed and non-inflamed skin, and induced DTH and protective responses to Leishmania challenge.

Contributed by Paula Hochman

ABSTRACT: Dermal resident memory CD4(+) T cells (dTrm) provide protection against vector-borne infections. However, the factors that promote their development remain unclear. We tested if an mRNA vaccine, encoding a protective leishmanial antigen, induced dTrm cells. The mRNA vaccine induced robust systemic T-cell responses, but few Trm cells were found in the skin. Since IL-12 promotes Th1 responses, we tested whether IL-12 mRNA combined with the mRNA vaccine could enhance dTrm cell development. This combination significantly expanded Leishmania-specific Th1 cells expressing skin-homing molecules and memory T cell markers in the draining lymph node. Additionally, higher numbers of dTrm cells were maintained in the skin, and mice exhibited functional immunity indicated by a delayed hypersensitivity response and protection upon challenge with Leishmania. These findings highlight IL-12 as a key driver of CD4(+) dTrm development, enabling their global seeding across the skin, and underscore the potential of IL-12-enhanced mRNA vaccines to generate durable immunity against cutaneous leishmaniasis and other skin-targeted infections.

Author Info: (1) Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA. (2) Department of Pathobiology, School of Veterinary Medicine, Uni

Author Info: (1) Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA. (2) Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA. (3) Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA. (4) Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA. (5) Department of Immunology, Max Rady College of Medicine, University of Manitoba, Winnipeg, MB, Canada. (6) Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA. (7) Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA. (8) Acuitas Therapeutics, Vancouver, BC, Canada. (9) Department of Immunology, Max Rady College of Medicine, University of Manitoba, Winnipeg, MB, Canada. (10) Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Penn Institute for RNA Innovation, University of Pennsylvania, Philadelphia, PA, USA. (11) Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA. (12) Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA. pscott@upenn.edu.

De novo-designed pMHC binders facilitate T cell-mediated cytotoxicity toward cancer cells Spotlight 

Johansen, Wolff, Scapolo, et al. used a known crystal structure, RFdiffusion, and other generative models to rapidly de novo design high-affinity minibinders (miBds) targeting the NY-ESO-1 peptide SllMWITQC on HLA-A*02:01. In silico cross-panning and molecular dynamics simulations allowed prescreening of specificity, which was later confirmed in vitro. The miBd structure was validated through cryo-electron microscopy, and when incorporated as a CAR on T cells, miBd binding induced killing of NY-ESO-1+ melanoma cells. The researchers also designed and validated a miBd to a neoantigen pMHC complex for which no experimental structure was available.

Contributed by Lauren Hitchings

Johansen, Wolff, Scapolo, et al. used a known crystal structure, RFdiffusion, and other generative models to rapidly de novo design high-affinity minibinders (miBds) targeting the NY-ESO-1 peptide SllMWITQC on HLA-A*02:01. In silico cross-panning and molecular dynamics simulations allowed prescreening of specificity, which was later confirmed in vitro. The miBd structure was validated through cryo-electron microscopy, and when incorporated as a CAR on T cells, miBd binding induced killing of NY-ESO-1+ melanoma cells. The researchers also designed and validated a miBd to a neoantigen pMHC complex for which no experimental structure was available.

Contributed by Lauren Hitchings

ABSTRACT: The recognition of intracellular antigens by CD8(+) T cells through T cell receptors (TCRs) is central for adaptive immunity against infections and cancer. However, the identification of TCRs from patient material remains complex. We present a rapid de novo minibinder (miBd) design platform leveraging state-of-the-art generative models to engineer miBds targeting the cancer-associated peptide-bound major histocompatibility complex (pMHC) SLLMWITQC/HLA-A*02:01 (NY-ESO-1). Incorporating in silico cross-panning enabled computational prescreening of specificity, and molecular dynamics simulations allowed for improved predictability of in vitro success. We identified a high-affinity NY-ESO-1 binder and confirmed its structure using cryo-electron microscopy, which, when incorporated in a chimeric antigen receptor, induced killing of NY-ESO-1(+) melanoma cells. We further designed and validated binders to a neoantigen pMHC complex, RVTDESILSY/HLA-A*01:01, with unknown structure, demonstrating the potential for precision immunotherapy.

Author Info: (1) Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark. (2) Department of Biotechnology and Biomedicine, Technical University of Denmark, Kon

Author Info: (1) Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark. (2) Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark. (3) Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark. Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark. (4) Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA. (5) Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark. Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark. (6) Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA. (7) Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark. (8) Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark. (9) Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark. (10) Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark. (11) Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark. (12) Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark. (13) Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark. (14) Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark. (15) Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA. (16) Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA. (17) Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark. (18) Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark. (19) Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark. (20) Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark. (21) Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark. (22) Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark. (23) Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark. (24) Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA. (25) Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark. (26) Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark. (27) Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark.

De novo design and structure of a peptide-centric TCR mimic binding module Spotlight 

Householder et al. used RFdiffusion and ProteinMPNN to de novo design an α-helical TCR mimic (TCRm) specific for an NY-ESO-1 peptide on HLA:A*02. This TCRm showed high specificity and affinity, with TCR-like docking that was strongly focused on the upward-facing peptide side chains. While a structure-informed in silico screen of 14,363 HLA-A*02 peptides (detected by immunopeptidomics) predicted two off-target peptides, when incorporated into a T cell engager, the TCRm showed peptide selectivity (in the 1–10 nM range) and supported T cell activation and cytotoxicity. The TCRm also supported antigen-specific T cell responses when incorporated into a CAR.

Contributed by Lauren Hitchings

Householder et al. used RFdiffusion and ProteinMPNN to de novo design an α-helical TCR mimic (TCRm) specific for an NY-ESO-1 peptide on HLA:A*02. This TCRm showed high specificity and affinity, with TCR-like docking that was strongly focused on the upward-facing peptide side chains. While a structure-informed in silico screen of 14,363 HLA-A*02 peptides (detected by immunopeptidomics) predicted two off-target peptides, when incorporated into a T cell engager, the TCRm showed peptide selectivity (in the 1–10 nM range) and supported T cell activation and cytotoxicity. The TCRm also supported antigen-specific T cell responses when incorporated into a CAR.

Contributed by Lauren Hitchings

ABSTRACT: T cell receptor (TCR) mimics offer a promising platform for tumor-specific targeting of peptide-major histocompatibility complex (pMHC) in cancer immunotherapy. In this study, we designed a de novo α-helical TCR mimic (TCRm) specific for the NY-ESO-1 peptide presented by human leukocyte antigen (HLA)-A*02, achieving high on-target specificity with nanomolar affinity (dissociation constant Kd = 9.5 nM). The structure of the TCRm-pMHC complex at 2.05-Å resolution revealed a rigid TCR-like docking mode with an unusual degree of focus on the up-facing NY-ESO-1 side chains, suggesting the potential for reduced off-target reactivity. Indeed, a structure-informed in silico screen of 14,363 HLA-A*02 peptides correctly predicted two off-target peptides, yet our TCRm maintained peptide selectivity and cytotoxicity as a T cell engager. These results represent a path for precision targeting of tumor antigens with peptide-focused α-helical TCR mimics.

Author Info: (1) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. Program in Immunology, Stanford University School of Medicine, Stanf

Author Info: (1) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. Program in Immunology, Stanford University School of Medicine, Stanford, CA, USA. (2) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (3) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (4) Department of Computer Science, Stanford University, Stanford, CA, USA. (5) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (6) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (7) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (8) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. (9) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA. (10) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA.

Nanobody-based bispecific antibody engagers targeting CTLA-4 or PD-L1 for cancer immunotherapy Featured  

Liu, Le Gall, Alexander, et al. developed bivalent nanobody conjugates targeting CTLA-4 or PD-L1, which could be loaded with small-molecule drug payloads. In mouse tumor models, the nanobody conjugates trafficked to the tumor and inhibited tumor growth. To improve the antitumor effects of the PD-L1 conjugate, maytansine and a STING agonist were assessed as payloads, which increased local T cell activation, reduced tumor growth, and improved survival.

Liu, Le Gall, Alexander, et al. developed bivalent nanobody conjugates targeting CTLA-4 or PD-L1, which could be loaded with small-molecule drug payloads. In mouse tumor models, the nanobody conjugates trafficked to the tumor and inhibited tumor growth. To improve the antitumor effects of the PD-L1 conjugate, maytansine and a STING agonist were assessed as payloads, which increased local T cell activation, reduced tumor growth, and improved survival.

ABSTRACT: As immune checkpoint blockade induces durable responses in only a subset of patients, more effective immunotherapies are needed. Here we present bispecific antibody engagers, fusion proteins composed of a nanobody that recognizes immunoglobulin kappa light chains (VHH(kappa)) and a nanobody that recognizes either CTLA-4 or PD-L1. These fusions show strong antitumour activity in mice through recruitment of polyclonal immunoglobulins independently of specificity or isotype. The anti-CTLA-4 VHH-VHH(kappa) conjugate demonstrates superior antitumour activity compared with the conventional monoclonal anti-CTLA-4 antibody and reduces the number of intratumoural regulatory T cells in a mouse model of colorectal carcinoma. The anti-PD-L1 VHH-VHH(kappa) conjugate is less effective in the colorectal carcinoma model while still outperforming a conventional antibody of similar specificity. The potency of the anti-PD-L1 VHH-VHH(kappa) conjugate was enhanced by installation of the cytotoxic drug maytansine or a STING agonist. The ability of such fusions to engage the Fc-mediated functions of all immunoglobulin isotypes is an appealing strategy to further improve on the efficacy of immune checkpoint blockade, commonly delivered as a monoclonal immunoglobulin of a single defined isotype.

Author Info: (1) Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA. (2) Program in Cellular and Molecular Medicine, Boston Children

Author Info: (1) Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA. (2) Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA. (3) Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA. (4) Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA. (5) Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA. (6) Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA. hidde.ploegh@childrens.harvard.edu.

Combination of pembrolizumab and radiotherapy induces systemic antitumor immune responses in immunologically cold non-small cell lung cancer Spotlight 

Huang, Theelen, and Belcaid et al. report the immunomodulatory effects of stereotactic body radiation therapy (SBRT) followed by pembrolizumab in patients with metastatic NSCLC based on a phase 2 trial. Multiomic analysis of serial tissue and blood biospecimens revealed upregulation of adaptive immunity programs, and longer PFS in immunologically cold tumors harboring features of immunotherapy resistance (TMB-low, PD-L1-null or Wnt-mutated) in the SBRT arm. Radioimmunotherapy upregulated interferon signaling, antigen presentation, and T cell infiltration in abscopal tumor sites, and induced systemic neoantigen-reactive T cell responses.

Contributed by Shishir Pant

Huang, Theelen, and Belcaid et al. report the immunomodulatory effects of stereotactic body radiation therapy (SBRT) followed by pembrolizumab in patients with metastatic NSCLC based on a phase 2 trial. Multiomic analysis of serial tissue and blood biospecimens revealed upregulation of adaptive immunity programs, and longer PFS in immunologically cold tumors harboring features of immunotherapy resistance (TMB-low, PD-L1-null or Wnt-mutated) in the SBRT arm. Radioimmunotherapy upregulated interferon signaling, antigen presentation, and T cell infiltration in abscopal tumor sites, and induced systemic neoantigen-reactive T cell responses.

Contributed by Shishir Pant

ABSTRACT: The abscopal effects of radiation may sensitize immunologically cold tumors to immune checkpoint inhibition. We investigated the immunostimulatory effects of radiotherapy leveraging multiomic analyses of serial tissue and blood biospecimens (n = 293) from a phase 2 clinical trial of stereotactic body radiation therapy (SBRT) followed by pembrolizumab in metastatic non-small cell lung cancer ( NCT02492568 ). Participants with immunologically cold tumors (low tumor mutation burden, null programmed death ligand 1 expression or Wnt pathway mutations) had significantly longer progression-free survival in the SBRT arm. Induction of interferon-γ, interferon-α and antigen processing and presentation gene sets was significantly enriched after SBRT in nonirradiated tumor sites. Significant on-therapy expansions of new and pre-existing T cell clones in both the tumor (abscopal) and the blood (systemic) compartments were noted alongside clonal neoantigen-reactive autologous T cell responses in participants with long-term survival after radioimmunotherapy. These findings support the systemic immunomodulatory and antitumor effects of radioimmunotherapy and may open a therapeutic window of opportunity to overcome immunotherapy resistance.

Author Info: (1) The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. (2) Netherlands Cancer Institute, Amsterdam, The Netherlands. (3

Author Info: (1) The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. (2) Netherlands Cancer Institute, Amsterdam, The Netherlands. (3) The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. (4) The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. (5) Netherlands Cancer Institute, Amsterdam, The Netherlands. (6) The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. The Bloomberg~Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, Baltimore, MD, USA. (7) The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. (8) The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. (9) The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. (10) The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. (11) The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Institute for Computational Medicine, Johns Hopkins University, Baltimore, MD, USA. (12) The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. (13) Department of Pulmonary Diseases, Radboud University Medical Center, Nijmegen, The Netherlands. (14) The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. (15) The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. The Bloomberg~Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, Baltimore, MD, USA. (16) Netherlands Cancer Institute, Amsterdam, The Netherlands. (17) The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA. vanagno1@jhmi.edu. The Bloomberg~Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, Baltimore, MD, USA. vanagno1@jhmi.edu.

ANV600 is a novel PD-1 targeted IL-2Rβγ agonist that selectively expands tumor antigen-specific T cells and potentiates PD-1 checkpoint inhibitor therapy

Spotlight 

Murer et al. engineered ANV600 – a novel bispecific antibody–cytokine fusion protein that binds to a non-blocking PD-1 epitope and targets IL-2 to PD-1-expressing cells, without interfering with concomitant anti-PD-1 therapy. ANV600 selectively expanded tumor antigen-specific CD8+ T cells while avoiding conventional IL-2 systemic toxicity and Treg expansion. ANV600 significantly inhibited tumor growth across multiple tumor models, and enhanced the efficacy of PD-1 checkpoint inhibitors. CD8+ T cell depletion, but not lymph node trafficking blockade, abrogated the therapeutic efficacy, suggesting that ANV600 efficacy is primarily driven by tumor-resident CD8+ T cells.

Contributed by Shishir Pant

Murer et al. engineered ANV600 – a novel bispecific antibody–cytokine fusion protein that binds to a non-blocking PD-1 epitope and targets IL-2 to PD-1-expressing cells, without interfering with concomitant anti-PD-1 therapy. ANV600 selectively expanded tumor antigen-specific CD8+ T cells while avoiding conventional IL-2 systemic toxicity and Treg expansion. ANV600 significantly inhibited tumor growth across multiple tumor models, and enhanced the efficacy of PD-1 checkpoint inhibitors. CD8+ T cell depletion, but not lymph node trafficking blockade, abrogated the therapeutic efficacy, suggesting that ANV600 efficacy is primarily driven by tumor-resident CD8+ T cells.

Contributed by Shishir Pant

Background: Combining interleukin-2 (IL-2) agonism with programmed cell death protein 1 (PD-1) checkpoint inhibition has shown synergistic potential in reinvigorating antitumor T cell responses. However, integrating these two mechanisms within a single molecule has been challenging due to competing requirements for PD-1 engagement and IL-2 receptor signaling. ANV600 is a novel bispecific antibody-cytokine fusion protein that targets a non-blocking epitope on PD-1, enabling cis-targeted IL-2Rβγ agonism while preserving combinability with therapeutic PD-1 inhibitors. This design allows for selective expansion of tumor antigen-specific T cells while avoiding the systemic toxicity and regulatory T cell (Treg) expansion associated with conventional IL-2 therapies.
Methods: The PD-1-targeting antibody used in ANV600 was generated by immunization of humanized mice and selected for its ability to bind PD-1 without blocking the binding epitope of PD-1 checkpoint blocking agents. ANV600 was evaluated in multiple syngeneic tumor models using human PD-1 transgenic mice. Tumor-infiltrating lymphocytes were analyzed to assess the selectivity of ANV600 for PD-1+ T cell subsets. Combination studies with pembrolizumab and nivolumab were performed to assess synergy with checkpoint inhibitors.
Results: ANV600 significantly inhibited tumor growth as monotherapy across multiple models, including the immune checkpoint-resistant B16F10 melanoma. By targeting PD-1, ANV600 selectively expanded tumor antigen-specific CD8+T cells, particularly progenitor exhausted (Tpex) and cytotoxic exhausted (Tcex) subsets, while sparing Tregs and NK cells. Combination with pembrolizumab and nivolumab resulted in additive effects, consistent with the complementary roles of PD-1 blockade in expanding Tpex cells and IL-2Rβγ signaling in reprogramming Tcex cells. ANV600's efficacy was dependent on CD8+T cells and primarily driven by tumor-resident T cells, as it remained effective despite blocked lymph node trafficking (FTY720) but was abrogated on CD8+ T cell depletion.
Conclusions: ANV600 represents a novel approach to delivering IL-2Rβγ agonism specifically to PD-1+ cells while preserving the binding site for PD-1 checkpoint inhibitors. By targeting a non-blocking epitope on PD-1, ANV600 enables the selective expansion of tumor-reactive CD8+ T cells while allowing independent and optimized dosing of both agents. This design ensures combinability with PD-1 inhibitors at clinically relevant doses, including in patients previously treated with checkpoint blockade. These findings support the clinical development of ANV600 as both a monotherapy and a combination therapy in cancer immunotherapy.

Author Info: (1) ANAVEON AG, Basel, Switzerland. (2) ANAVEON AG, Basel, Switzerland. (3) ANAVEON AG, Basel, Switzerland. (4) ANAVEON AG, Basel, Switzerland. (5) ANAVEON AG, Basel, Switzerland.

Author Info: (1) ANAVEON AG, Basel, Switzerland. (2) ANAVEON AG, Basel, Switzerland. (3) ANAVEON AG, Basel, Switzerland. (4) ANAVEON AG, Basel, Switzerland. (5) ANAVEON AG, Basel, Switzerland. (6) ANAVEON AG, Basel, Switzerland. (7) ANAVEON AG, Basel, Switzerland. (8) ANAVEON AG, Basel, Switzerland. (9) ANAVEON AG, Basel, Switzerland. (10) ANAVEON AG, Basel, Switzerland. (11) ANAVEON AG, Basel, Switzerland christoph.huber@anaveon.com.

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