Abstract The KRASG12D mutation is an ideal target for anti-cancer therapies as its expression is typically clonal, restricted to cancer tissue, and is among the most common oncogenic drivers in solid tumors. TCR-T cell therapies have demonstrated clinical activity in some solid cancers but have been limited by heterogeneous antigen expression and unfavorable tumor microenvironments. By targeting the KRASG12D mutation for which the cancer has established genetic dependency, AFNT-212 is designed to selectively target all cancer cells while avoiding on-target/off-tumor toxicities. AFNT-212 is non-virally engineered to knock-in a 5-transgene cassette expressing a high-avidity TCR specific for the KRASG12D mutation, a CD8α/β coreceptor, and a chimeric cytokine receptor. Transgene insertion at the TRAC locus disrupts expression of the endogenous TCRα, further enhancing the expression/activity of the transgenic KRASG12D TCR. Primary human CD8+ and CD4+ T cells were genetically engineered by a novel CRISPR-Cas nuclease system to integrate AFNT-212 transgenes within the TRAC locus. A cGMP compatible scale-up process for non-viral knock-in was established to support AFNT-212 clinical manufacturing. The activity of AFNT-212 was assessed against a panel of human KRASG12D tumor cell lines in vitro and established mouse xenograft models in vivo. The preclinical safety profile of AFNT-212 was evaluated by X-scan and crossreactivity assessment, alloreactivity studies, and cytokine independent growth studies. The specificity of gene-editing (GE) was assessed by an unbiased oligo-capture method followed by targeted sequencing. AFNT-212 TCR-T cells demonstrated potent in vitro anti-tumor activity against endogenously expressing HLA-A*11:01 KRASG12D tumor cells, including during chronic exposure to viable tumor cells. AFNT-212 TCR-T cells showed robust antitumor activity in established xenograft mouse models in vivo. No cross-reactivity was identified for the KRASG12D TCR against potential self-peptides even at supraphysiological levels, demonstrating high specificity of the TCR. No alloreactivity or cytokine-independent proliferation was observed. GE safety evaluations did not reveal any off-target activity using high sensitivity (~0.1%) NGS-based analyses or any GE-associated chromosomal rearrangements. The manufacturing of AFNT-212 consistently delivered >50-fold expansion of engineered TCR-T cells to meet expected clinical dose levels and exhibit memory/stemness phenotypes and negligible markers of immunologic exhaustion. AFNT-212, a novel TCR T cell therapy targeting KRASG12D mutant tumors, demonstrates robust activity against KRASG12D mutant tumors in vitro and in vivo. The robust manufacturing process developed using non-viral gene editing in the TRAC locus will support future clinical development of AFNT-212. Citation Format: Allison Drain, Nicholas Rouillard, Nathaniel Swanson, Martina Canestraro, Santosh Narayan, Tyler Warner, Nicole Danek, Ken Gareau, Jinsheng Liang, Luhua Shen, Tanya Tetrault, Iqraa Priyata, Sarah Vidyasagar, Taylor Riggins-Walker, Hui-Wen Liu, Klaus Pechhold, Lauren Brown, Joshua Francis, Xingyue He, Patrick Browne, Rebecca Lamothe, Meghan Storlie, Gregory Cost, Thomas M. Schmitt, Philip D. Greenberg, Smita S. Chandran, Christopher A. Klebanoff, Hubert Lam, Ankit Gupta, Damien Hallet, Gary Shapiro, Kim Nguyen, Loïc Vincent. AFNT-212: A TRAC-knocked-in KRASG12D-specific TCR-T cell product enhanced with CD8αβ and a chimeric cytokine receptor for treatment of solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 9.

Abstract Adoptive T cell therapy (ACT) has demonstrated antitumor efficacy in patients with solid cancers but requires further optimization to become a reproducibly effective treatment. T cell receptor (TCR)-engineered T cells recognize peptides derived from intracellular and surface proteins presented in the context of MHC class I. Targeting mutated oncogenic drivers addresses many of the major obstacles of this modality, in that the antigenic epitope is: 1) tumor-specific, 2) essential for tumor survival, and 3) derived from a stably expressed protein. However, the immune-suppressive tumor microenvironment makes further optimization of engineered T cells necessary to bring long-term clinical benefit to patients. For an optimal anti-tumor response, T cells require three signals: TCR, co-stimulation, and cytokine signaling. The tumor suppressor TP53 is the most frequently mutated gene across human cancers, with a highly recurrent arginine to histidine hotspot alteration in codon 175 leading to novel tumor-dependent functions. Here we report the use of a novel CRISPR-Cas nuclease system to knock-in a six-parameter multi-cistronic cassette into the TRAC locus with high efficiency. We employed several strategies to maximize the potency and durability of a TCR-T cell product targeting the p53 R175H oncogenic driver, including: 1) A high-affinity TCR (α and β chains) specific for the p53 R175H mutation presented by HLA-A*02:01 permits the recognition of tumor cells expressing even low levels of the epitope (Signal 1), 2) Inclusion of the CD8αβ co-receptor drives stimulation of CD4+ T cells with the MHC class I restricted TCR, allowing for a physiologic coordinated immune response required for maximal efficacy, 3) A FAS-41BB switch receptor acts as a dominant negative to the FASL-inducing apoptotic signal in the tumor microenvironment and drives stimulation and persistence of the T cell product via 41BB co-stimulatory signaling (Signal 2), 4) A chimeric cytokine receptor (constitutive Interleukin Receptor) promotes expansion and survival while avoiding immunologic exhaustion (Signal 3). Together, these strategies deliver the three signals required for maximal T cell function: antigen-driven activation, co-stimulation, and growth/survival-promoting cytokine signaling. The non-viral TRAC-knocked-in T cells demonstrate robust and specific cytotoxicity against endogenously expressing HLA-A*02:01 and p53 R175H cell lines in vitro and effective anti-tumor activity in vivo while maintaining a favorable preclinical safety profile. These data support the planned clinical development of a novel non-viral TRAC-knocked-in T cell therapy for the treatment of p53 R175H-mutant solid tumors. Citation Format: Santosh Narayan, Ken Gareau, Ankit Gupta, Josh Ferrell, Nicholas Rouillard, Tyler Warner, Jinsheng Liang, Luhua Shen, Tanya Tetrault, Joshua Francis, Xingyue He, Patrick J. Browne, Rebecca Lamothe, Meghan D. Storlie, Gregory J. Cost, Thomas M. Schmitt, Philip D. Greenberg, Smita S. Chandran, Damien Hallet, Michael Gormally, Chistopher A. Klebanoff, Gary Shapiro, Kim Nguyen, Loïc Vincent. Non-viral engineered T cell therapy specific for the hotspot mutation p53 R175H that integrates signal 1 (TCR), signal 2 (co-stimulation) and signal 3 (cytokine) and co-opts FasL-dependent apoptosis to achieve a coordinated antitumor CD4/8 T cell response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 10.

Background Adoptive T cell therapy has demonstrated clinical activity in a subset of patients with solid tumors; however, consistent responses will require further optimization. T cell receptor (TCR)-engineered T cells recognize peptides derived from intracellular and surface proteins presented in the context of MHC class I. Immunologic targeting of recurrently mutated oncogenic drivers, such as KRAS, overcomes many of the major obstacles of this modality because the resulting epitope is: 1) tumor-specific, 2) essential for cancer cell fitness, and 3) derived from a stably expressed non-self protein. AFNT-212 is a next-generation engineered T cell therapy that uses non-viral targeted knock-in (KI) at the TCRα constant (TRAC) locus to express a multi-cistronic cassette that includes 1) a high-affinity TCR specific for KRASG12D mutation, 2) a CD8αβ coreceptor, and 3) a chimeric cytokine receptor. Methods Human CD4+ and CD8+ T cells were genetically engineered by a novel CRISPR-Cas nuclease and gRNAs targeting TRAC and the TCRβ constant (TRBC) genes allowing for knock-out of the endogenous TCR loci and simultaneous integration of the non-viral plasmid-based transgene cassette. Engineered T cells were assessed for specificity and potency, including activation, proliferation, and cytotoxicity, against KRAS G12D peptide presented by HLA-A*11:01 and a panel of KRAS G12D-expressing tumor cell lines. In vitro safety studies were performed along with in vivo efficacy studies in multiple human xenograft models. Results Engineered primary T cells showed specific recognition of KRAS G12D peptide, demonstrated cytotoxicity against endogenously expressing HLA-A*11:01+/KRAS G12D+ cell lines in tumor cell re-challenge assays in vitro, and mediated robust anti-tumor activity in vivo. Inclusion of the chimeric cytokine receptor allowed for a more potent anti-tumor response stemming from improved T cell expansion and resistance to exhaustion. No off-target liabilities were identified upon co-incubation of AFNT-212 with all possible peptides in the human proteome matching the xScan-defined epitope recognition motif for the TCR, demonstrating specificity. Gene editing safety evaluation did not reveal any off-target activity for the CRISPR-Cas nucleases and engineered T cells did not show cytokine-independent proliferation, collectively supporting a favorable pre-clinical safety profile for AFNT-212. Conclusions We report a novel TCR gene therapy approach targeting mutant KRAS G12D-expressing tumors with a coordinated CD4/CD8 T cell response that has a promising efficacy and safety profile. Our work supports the planned clinical development of AFNT-212 as a novel non-viral KI TCR-engineered T cell therapy for KRAS-mutant solid tumors.

2543 Background: KRAS is the most common oncogenic driver mutation in solid tumors, promoting the initiation and progression of many uncurable cancers, including colorectal, pancreatic and lung cancer. While small molecule inhibitors to KRAS G12C mutations have been approved, there are no targeted therapies available for patients with highly prevalent KRAS G12V mutations. TCR-T cell therapies have demonstrated remarkable responses in clinical trials, but their durability has been limited by the immunosuppressive tumor microenvironment (TME). AFNT-211 is an autologous T cell therapy engineered to express an HLA-A*11:01 KRAS G12V -specific TCR, further enhanced with CD8α/β coreceptor and a FAS-41BB switch receptor to drive T cell persistence and durable clinical responses. CD8α/β coreceptor enables a coordinated CD4+/CD8+ T cell response and FAS-41BB converts the FAS ligand (FASL) TME death signal into a costimulatory signal through 41BB activation. Methods: AFNT-211 was assessed for efficacy in vitro against a panel of KRAS G12V -expressing tumor cell lines and in vivo using human xenograft mouse models. In vitro safety studies were performed to assess potential cross-reactivity, alloreactivity, and cytokine-independent growth. The clinical manufacturing process consists of autologous CD4+/CD8+ T cells transduced with lentivirus and expanded using culture conditions that drive robust expansion while preserving stem-like properties. Phenotypic and functional analyses of AFNT-211 were performed using flow cytometry and cell-based assays. Results: Coculture of AFNT-211 with a panel of KRAS G12V -expressing tumor cell lines led to significant effector cytokine secretion, T cell proliferation, and tumor cell killing. The CD8α/β coreceptor enabled CD4+ T cell recognition of KRAS G12V and greater overall cytotoxicity. The FAS-41BB switch receptor dramatically augmented the magnitude and durability of the anti-tumor response against FASL-expressing tumor cells. XScan mutagenesis and potential off-target peptide testing revealed no significant cross-reactivities. No alloreactivity was observed against a panel of lymphoblastoid cell lines presenting the most frequent HLA types in the US population. Potent anti-tumor response was observed in vivo in a mouse xenograft model. Affini-T’s Thrive manufacturing platform used for the production of AFNT-211 consistently delivers >30-40e 9 TCR-engineered T cells with a high frequency of naïve and central memory T cells expressing negligible markers of exhaustion. Conclusions: The AFNT-211 manufacturing process generates a large number of TCR-T cells with desirable stem-like properties optimized for clinical dosing. Preclinical data demonstrated a potent and safe profile for AFNT-211 that supports clinical development in HLA-A*11:01 patients with advanced/metastatic solid tumors harboring a KRAS G12V mutation.

Background The AFNT-212 cell therapy consists of autologous CD8+ and CD4+ T cells expressing 1) a T Cell Receptor (TCR) specific for the prevalent oncogenic driver KRAS G12D mutation presented by HLA-A*11:01, 2) a chimeric cytokine receptor, and 3) the CD8α/β coreceptor enabling a coordinated CD4+/CD8+ anti-tumor response to promote T cell activity while minimizing exhaustion. While viral vectors, including lentivirus (LVV), have been a standard modality to deliver transgenes for cell therapies, they are limited by cargo size and manufacturing complexity. In comparison, gene-editing mediated targeted knock-in (KI) of a non-virally delivered transgene cassette overcomes limitations of LVV-mediated delivery and improves function and safety profile of the engineered cells. Methods Human CD4+ and CD8+ T cells from healthy volunteers or patients were engineered by a novel CRISPR-Cas nuclease and gRNAs targeting the T-cell receptor α constant (TRAC) and T-cell receptor β constant (TRBC) genes to knock-out the endogenous TCR and simultaneously integrate a non-viral plasmid-based transgene cassette. Human CD4+ and CD8+ T cells were engineered in parallel with lentivirus encoding the same transgene cassette and CRISPR-Cas targeting TRAC and TRBC genes to knock-out endogenous TCR. Engineered T cells were assessed via KRAS G12D peptide stimulation and co-culture with KRAS G12D-expressing tumor cells for in vitro activation and cytotoxicity. In vitro safety studies were performed and in vivo efficacy studies were conducted using human KRAS G12D xenografts in NSG mice. Results Non-viral KI generated lower vector copy number per cell than LVV but drove higher transgene expression, suggesting the EF1α promoter within the KI construct outperforms the MSCV promoter of the LVV. T cells engineered with either non-viral KI or LVV demonstrated specific and sensitive recognition of the target KRAS G12D peptide. However, KI-engineered cells demonstrated improved cytotoxicity against endogenously-expressing HLA-A*11–01 and KRAS G12D cell lines in tumor cell rechallenge assays in vitro. KI-engineered cells also showed superior anti-tumor activity in established subcutaneous tumor bearing mice. Off-target assessment was similar for the KI and LVV products, as identified by co-incubation with all possible peptides in the human proteome matching the TCR recognition motif. Optimized KI process generated large number of transgenic cells with naïve and memory phenotype potentially suitable for clinical applications. Conclusions Non-viral targeted KI engineered AFNT-212 cells drive a robust coordinated CD4/CD8 T cell response against KRAS G12D-harboring tumors and outperform LVV-engineered cells. Our work supports the planned clinical development of this novel TCR-engineered T cell therapy for treating KRAS-mutant solid tumors. Ethics Approval These studies were approved by Affini-T Therapeutics and Explora Biolabs' Institutional Animal Care and Use Committee, approval number EB17–010-303.

Mutations in the RAS family are highly prevalent in human cancers, including up to 35% of non-small cell lung, 45% of colorectal, and 95% of pancreatic cancers. Kirsten rat sarcoma viral oncogene homologue (KRAS) is the most frequently mutated RAS oncogene and patients with KRAS mutations have poor responses to standard treatment regimens. Therefore, successful KRAS-targeted therapies represent a high unmet need. The AFNT-111 cellular therapy consists of autologous CD8+ and CD4+ T cells expressing a high affinity TCR specific for the prevalent KRASG12V mutation presented by HLA-A*11:01, one of the most common HLA alleles worldwide. AFNT-111 is engineered to express the CD8α/β coreceptor, enabling a coordinated CD4+/CD8+ tumor response to induce robust T cell activity and persistence while minimizing T cell exhaustion. Lentiviral vector was used to transduce primary healthy human CD4+ and CD8+ T cells with the KRASG12V-specific human TCR and CD8α/β coreceptor. Engineered T cells were assessed against exogenous KRASG12V peptide as well as a panel of tumor cell lines endogenously expressing KRASG12V and probed for in vitro activation, proliferation, cytotoxicity and cytokine secretion. In parallel, in vitro safety studies were performed to evaluate autoantigen cross-reactivity and alloreactivity. In vivo efficacy studies were conducted using established human KRASG12V xenografts in NSG mice. AFNT-111 demonstrated potent functional avidity towards the KRASG12V peptide with no reactivity against KRASWT. Several naturally expressing KRASG12V human tumor cell lines led to significant AFNT-111 T cell activation and proliferation, as well as potent cytotoxicity. The addition of the CD8α/β coreceptor revealed a coordinated and synergistic cytotoxic CD4+ and CD8+ T cell response, enhancing activation of the AFNT-111 cell product. Robust cytotoxicity was associated with increased T cell secretion of IFNγ and TNFα. Durable in vivo anti-tumor efficacy was observed in three established mouse xenograft tumor models. XScan studies using amino acid substitutions of the index KRASG12V peptide revealed a restrictive TCR recognition motif. Further, potentially cross-reactive human self-peptides matching this motif were tested and no significant cross-reactivities were identified. To test alloreactivity, a large lymphoblastoid cell line library was assessed with no alloreactive responses detected. A streamlined manufacturing process has also been developed in which CD4+/CD8+ T cell ratios are controlled, with the final AFNT-111 product retaining a high degree of stem-like properties. AFNT-111 preclinical data demonstrate a highly potent and specific TCR-engineered T cell product that is cytotoxic to KRASG12V-expressing tumor cells both in vitro and in vivo. Cross-reactivity and alloreactivity assessments establish a potentially favorable safety profile of AFNT-111, supporting clinical translation. First-in-human clinical studies will focus on advanced or metastatic pancreatic, colorectal, and lung cancer indications. Citation Format: Michele Hoffmann, Xingyue He, Cheryl Black, Joshua Francis, James Parsons, Christian Roy, Jinsheng Liang, Hongjing Qu, Martin Campbell, Tomasz Sewastianik, Jessica Webb, Aude Chapuis, Thomas Schmitt, Philip Greenberg, Damien Hallet, Markus Vallaster, Piotr Pierog, Gary Shapiro, Hubert Lam, Loic Vincent. AFNT-111: A novel TCR-engineered T cell therapy targeting the oncogenic driver KRAS G12V [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A009.

Background Mutations in the RAS family of genes are responsible for approximately 30% of all human cancers. Mutated RAS proteins are truncal oncogenic driver antigens essential for cancer development and progression making them optimal targets for cancer therapies by limiting tumor escape. The AFNT-111 cell therapy consists of autologous CD8+ and CD4+ T cells expressing a TCR specific for the highly prevalent KRASG12V mutation presented by HLA-A*11:01, one of the most common HLA alleles worldwide. AFNT-111 is also engineered to express the CD8α/β coreceptor, enabling a coordinated CD4+/CD8+ tumor response that aims to promote increased T cell activity and persistence while minimizing T cell exhaustion. Methods Lentiviral vector was used to transduce primary human CD4+ and CD8+ T cells with the KRASG12V-specific TCR and CD8α/β coreceptor. Engineered T cells were assessed against KRASG12V peptide and a panel of KRASG12V-expressing tumor cell lines for in vitro activation, proliferation, and cytotoxicity. In vitro safety studies were performed to evaluate self-peptide cross-reactivity and alloreactivity and in vivo efficacy studies were conducted using human KRASG12V xenografts in NSG mice. Results AFNT-111 demonstrated potent functional avidity for KRASG12V peptide with no reactivity to wildtype KRAS. Several naturally expressing KRASG12V human tumor cell lines, derived from lung, colorectal, and pancreatic cancer, triggered significant AFNT-111 T cell activation and proliferation, and potent cytotoxicity towards tumor cells. In vitro killing by AFNT-111 was consistently observed even after repeated tumor cell challenge. Robust in vivo anti-tumor efficacy was also observed in two established mouse xenograft tumor models. XScan studies using amino acid substitutions of the reference KRASG12V peptide revealed a restrictive TCR recognition motif limiting risk of promiscuous off-target activation. Further, potentially cross-reactive self-peptides in the human proteome matching this motif were tested and no cross-reactivities with significant avidity were identified. A large lymphoblastoid cell line library covering >95% of the most common HLA alleles was assessed with no alloreactive responses detected. For clinical studies, a robust manufacturing process has been developed in which CD4+/CD8+ T cell ratios are controlled, and the final AFNT-111 drug product preserves stem-like properties. Conclusions AFNT-111 preclinical data demonstrate a highly potent and specific TCR-engineered T cell product that is cytotoxic to KRASG12V-expressing tumor cells both in vitro and in vivo. Cross-reactivity and alloreactivity assessments established a strong safety profile of AFNT-111, supporting clinical translation. First-in-human clinical studies will focus on advanced or metastatic pancreatic, colorectal, and lung cancer indications. Ethics Approval These studies were approved by Affini-T Therapeutics and Fred Hutchinson Cancer Research Center Ethics Boards, approval number EB17-010-303 and PROTO000050898, respectively.

TPS9596 Background: Merkel cell carcinoma (MCC) is a highly aggressive skin cancer, with an incidence that has doubled in the last 20 years to approximately 3000 cases/year in the US. Over one-third of patients will develop widespread disease, and survival in these patients has been historically poor with a 5-year survival rate of < 10%. MCC is highly immune-sensitive due to the antigenicity of the cancer-causing Merkel cell polyomavirus (MCPyV) expressed in most MCC tumors. Although immune checkpoint inhibitors (ICIs) targeting the PD-(L)1 axis show promising efficacy, most MCC patients will eventually relapse. There is no standard of care for patients that become refractory to ICIs. We hypothesize that cellular immune therapies targeting MCPyV may provide additional clinical benefit to these patients. To test this, we have engineered high-affinity TCR T cells against MCPyV and initiated a clinical trial. Methods: NCT03747484 is an ongoing phase I/II, open label, investigator-initiated trial (IIT) of FH-MCVA2TCR in combination with an anti-PD-(L)1 checkpoint inhibitor and an agent to upregulate MHC-I expression on tumor cells. The trial is conducted in patients aged 18 years or older with metastatic or unresectable, histologically confirmed virus-positive MCC whose disease has progressed on or after treatment with a PD-(L)1 axis checkpoint inhibitor. Patients undergo leukapheresis to collect white blood cells (WBCs) for TCR T cell product manufacturing. The cell product is administered on day 0. Patients receive an agent to upregulate MHC-I on tumor cells and continue on an anti-PD-(L)1 checkpoint inhibitor for up to one year. In phase I, three patients receive up to two infusions of dose level 1 of the T cell product. The primary objectives of phase I are to determine safety and tolerability based on dose-limiting toxicities (DLT) during an observation period of 28 days after the first infusion. Phase II enrolls patients at dose level 2. The total sample size of phase II is 12 patients. The first three patients are enrolled in a staggered manner and observed for DLTs. If no DLTs occur, the remaining sample size is accrued in open enrollment. The main objectives of phase II are safety based on number of adverse events and preliminary efficacy based on tumor response according to RECISTv1.1 and iRECIST. Secondary/exploratory objectives include cellular kinetics of TCR T cells, T cell phenotype, tumor infiltration kinetics, and MHC-I expression dynamics over time. After the first 12 months, patients transition to a long-term follow-up (LTFU) study for up to 15 years as per FDA guidelines. Clinical trial information: NCT03747484.