High Functional Avidity Research Articles

CARs derived from broadly neutralizing, human monoclonal antibodies identified by single B cell sorting target hepatitis B virus-positive cells.

To design new CARs targeting hepatitis B virus (HBV), we isolated human monoclonal antibodies recognizing the HBV envelope proteins from single B cells of a patient with a resolved infection. HBV-specific memory B cells were isolated by incubating peripheral blood mononuclear cells with biotinylated hepatitis B surface antigen (HBsAg), followed by single-cell flow cytometry-based sorting of live, CD19+ IgG+ HBsAg+ cells. Amplification and sequencing of immunoglobulin genes from single memory B cells identified variable heavy and light chain sequences. Corresponding immunoglobulin chains were cloned into IgG1 expression vectors and expressed in mammalian cells. Two antibodies named 4D06 and 4D08 were found to be highly specific for HBsAg, recognized a conformational and a linear epitope, respectively, and showed broad reactivity and neutralization capacity against all major HBV genotypes. 4D06 and 4D08 variable chain fragments were cloned into a 2nd generation CAR format with CD28 and CD3zeta intracellular signaling domains. The new CAR constructs displayed a high functional avidity when expressed on primary human T cells. CAR-grafted T cells proved to be polyfunctional regarding cytokine secretion and killed HBV-positive target cells. Interestingly, background activation of the 4D08-CAR recognizing a linear instead of a conformational epitope was consistently low. In a preclinical model of chronic HBV infection, murine T cells grafted with the 4D06 and the 4D08 CAR showed on target activity indicated by a transient increase in serum transaminases, and a lower number of HBV-positive hepatocytes in the mice treated. This study demonstrates an efficient and fast approach to identifying pathogen-specific monoclonal human antibodies from small donor cell numbers for the subsequent generation of new CARs.

Abstract 8: Deep Immunomics pipeline for discovery and validation of novel cancer-specific T cell receptors

Abstract Background: T cell receptor-engineered T cell (TCR-T) therapy has been studied as a high potential approach for cancer treatment. Beyond cell-surface tumor antigens, TCR-Ts can recognize HLA-presented intracellular epitopes which allows them to address a wide range of cancer-specific targets. Existing TCR-T therapies focus on a limited number of epitopes, many of which are restricted to HLA allele HLA-A*02:01. Identification of novel anti-tumor TCRs covering a broad range of HLA alleles is necessary to address unmet clinical needs. Natural T cells undergo thymic selection and present a favorable safety profile for mining such anti-tumor TCRs for therapeutic development. However, discovery of natural anti-tumor TCRs has been challenging due to i) the absence of high-throughput methods for identifying tumor-reactive TCRs in limited quantity human samples and ii) lack of sensitivity in detecting low frequency, naturally occurring tumor-reactive T cells. Methods: We developed TargetScape® as a part of our Deep Immunomics platform for efficient discovery of natural tumor-specific TCRs in healthy donors and cancer patients. Cells from blood and tissue samples were stained with metal-barcoded peptide-MHC tetramers, containing 500+ tumor-specific antigens, alongside metal-conjugated antibodies, allowing rapid identification and high dimensional phenotypic analysis of CD8+ T cells at the millions of cells scale. Single-cell sequencing revealed paired TCR sequences. Selected TCRs were introduced into luminescent Jurkat reporter cells for validation screening with peptide-pulsed targets and the relevant HLA restriction. TCRs of interest were further integrated into primary T cells and evaluated for their effector function. Results: We utilize our sensitive, high-throughput TargetScape® platform to detect and characterize putative cancer-specific TCRs from blood and tissue samples in frequencies as low as ~0.003% of total CD8+ cells. We have functionally validated over 100 TargetScape® derived TCRs with luminescent Jurkat reporters. Validated TCRs cover high prevalence HLA alleles and target a broad range of cancer antigen classes, including tumor associated antigens (TAA), Human endogenous retroviruses (HERVs), shared splice variants, and frameshift mutations. Several natural TCRs demonstrate recognition of low concentrations of peptide, indicating high functional avidity. Further evaluations of specificity and activity of TCRs of interest transduced into primary T cells are currently in progress. Conclusions: Our Deep Immunomics platform allows us to identify and confirm activity of novel, naturally-derived TCR candidates against therapeutically relevant cancer antigens presented on six common HLA alleles. These findings validate our discovery workflow leading to a portfolio of potential clinically relevant candidates that are currently under preclinical development. Citation Format: Hannah Fields, Juliana Velez Lujan, Kan Xing Wu, Natalie Epstein, Nazihah Beevi, Florence Chioh, Nicholas Tan, Neeraja Kulkarni, Noella Mukobo, Srikanthi R, Yovita Purwanti, Evan Newell, Alessandra Nardin, Michael Fehlings, Katja Fink, Daniel MacLeod. Deep Immunomics pipeline for discovery and validation of novel cancer-specific T cell receptors [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 8.

Structural basis for self-discrimination by neoantigen-specific TCRs

T cell receptors (TCR) are pivotal in mediating tumour cell cytolysis via recognition of mutation-derived tumour neoantigens (neoAgs) presented by major histocompatibility class-I (MHC-I). Understanding the factors governing the emergence of neoAg from somatic mutations is a major focus of current research. However, the structural and cellular determinants controlling TCR recognition of neoAgs remain poorly understood. This study describes the multi-level analysis of a model neoAg from the B16F10 murine melanoma, H2-Db/Hsf2 p.K72N68-76, as well as its cognate TCR 47BE7. Through cellular, molecular and structural studies we demonstrate that the p.K72N mutation enhances H2-Db binding, thereby improving cell surface presentation and stabilizing the TCR 47BE7 epitope. Furthermore, TCR 47BE7 exhibited high functional avidity and selectivity, attributable to a broad, stringent, binding interface enabling recognition of native B16F10 despite low antigen density. Our findings provide insight into the generation of anchor-residue modified neoAg, and emphasize the value of molecular and structural investigations of neoAg in diverse MHC-I contexts for advancing the understanding of neoAg immunogenicity.

An EBV-related CD4 TCR immunotherapy inhibits tumor growth in an HLA-DP5+ nasopharyngeal cancer mouse model.

Adoptive transfer of T cell receptor-engineered T cells (TCR-T) is a promising strategy for immunotherapy against solid tumors. However, the potential of CD4+ T cells in mediating tumor regression has been neglected. Nasopharyngeal cancer is consistently associated with EBV. Here, to evaluate the therapeutic potential of CD4 TCR-T in nasopharyngeal cancer, we screened for CD4 TCRs recognizing EBV nuclear antigen 1 (EBNA1) presented by HLA-DP5. Using mass spectrometry, we identified EBNA1567-581, a peptide naturally processed and presented by HLA-DP5. We isolated TCR135, a CD4 TCR with high functional avidity, that can function in both CD4+ and CD8+ T cells and recognizes HLA-DP5-restricted EBNA1567-581. TCR135-transduced T cells functioned in two ways: directly killing HLA-DP5+EBNA1+ tumor cells after recognizing EBNA1 presented by tumor cells and indirectly killing HLA-DP5-negative tumor cells after recognizing EBNA1 presented by antigen-presenting cells. TCR135-transduced T cells preferentially infiltrated into the tumor microenvironment and significantly inhibited tumor growth in xenograft nasopharyngeal tumor models. Additionally, we found that 62% of nasopharyngeal cancer patients showed 50%-100% expression of HLA-DP on tumor cells, indicating that nasopharyngeal cancer is well suited for CD4 TCR-T therapy. These findings suggest that TCR135 may provide a new strategy for EBV-related nasopharyngeal cancer immunotherapy in HLA-DP5+ patients.

Selection of highly responsive T cell receptors by an analysis combining the expression of multiple markers.

The clinical success of T cell receptor (TCR) gene-transduced T (TCR-T) cell therapy is expected as one of the next-generation immunotherapies for cancer, in which the selection of TCRs with high functional avidity (high-functional TCRs) is important. One widely used approach to select high-functional TCRs is a comparison of the EC50 values of TCRs, which involves laborious experiments. Therefore, the establishment of a simpler method to select high-functional TCRs is desired. We herein attempted to establish a simple method to select high-functional TCRs based on the expression of T cell activation markers using the mouse T cell line BW5147.3 (BW). We examined relationships between the EC50 values of TCRs in interleukin-2 production and the expression levels of TCR activation markers on BW cells. In TCR-expressing BW cells stimulated with antigenic peptides, the CD69, CD137, and PD-1 expression was differentially induced by various doses of peptides. An analysis of TCRs derived from the tumor-infiltrating lymphocytes of murine melanoma and peripheral blood T cells of hepatocellular carcinoma patients treated with a peptide vaccination revealed that an analysis combining CD69, CD137, and PD-1 expression levels in BW cells stimulated with a single dose of an antigenic peptide selected high-functional TCRs with functional avidity assessed by EC50 values. Our method facilitates the section of high-functional TCRs among tumor-reacting TCRs, which will promote TCR-T cell therapy. The stimulation of BW cells expressing objective TCRs with a single dose of antigenic peptides and analysis combining the expression of CD69, CD137, and PD-1 allows us to select highly responsive TCRs.

Structural insights into protection against a SARS-CoV-2 spike variant by T cell receptor diversity

T cells play a crucial role in combatting SARS-CoV-2 and forming long-term memory responses to this coronavirus. The emergence of SARS-CoV-2 variants that can evade T cell immunity has raised concerns about vaccine efficacy and the risk of reinfection. Some SARS-CoV-2T cell epitopes elicit clonally restricted CD8+ T cell responses characterized by T cell receptors (TCRs) that lack structural diversity. Mutations in such epitopes can lead to loss of recognition by most T cells specific for that epitope, facilitating viral escape. Here, we studied an HLA-A2-restricted spike protein epitope (RLQ) that elicits CD8+ T cell responses in COVID-19 convalescent patients characterized by highly diverse TCRs. We previously reported the structure of an RLQ-specific TCR (RLQ3) with greatly reduced recognition of the most common natural variant of the RLQ epitope (T1006I). Opposite to RLQ3, TCR RLQ7 recognizes T1006I with even higher functional avidity than the WT epitope. To explain the ability of RLQ7, but not RLQ3, to tolerate the T1006I mutation, we determined structures of RLQ7 bound to RLQ-HLA-A2 and T1006I-HLA-A2. These complexes show that there are multiple structural solutions to recognizing RLQ and thereby generating a clonally diverse T cell response to this epitope that assures protection against viral escape and T cell clonal loss.

Identification of novel HLA-A*11:01-restricted HPV16 E6/E7 epitopes and T-cell receptors for HPV-related cancer immunotherapy

BackgroundE6 and E7 oncoproteins are considered ideal antigens of T cell therapy for human papillomavirus (HPV)-related cancers. However, little is known about the epitopes of E6 and E7 presented by...

Targeting NKG2A to boost anti-tumor CD8 T-cell responses in human colorectal cancer

Recently, the inhibitory CD94/NKG2A receptor has joined the group of immune checkpoints (ICs) and its expression has been documented in NK cells and CD8+ T lymphocytes in several cancers and some infectious diseases. In colorectal cancer (CRC), we previously reported that NKG2A+ tumor-infiltrating lymphocytes (TILs) are predominantly CD8+ αβ T cells and that CD94 overexpression and/or its ligand HLA-E were associated with a poor prognosis. This study aimed to thoroughly characterize the NKG2A+ CD8+ TIL subpopulation and document the impact of NKG2A on anti-tumor responses in CRC. Our findings highlight new features of this subpopulation: (i) enrichment in colorectal tumors compared to paired normal colonic mucosa, (ii) their character as tissue-resident T cells and their majority terminal exhaustion status, (iii) co-expression of other ICs delineating two subgroups differing mainly in the level of NKG2A expression and the presence of PD-1, (iv) high functional avidity despite reduced proliferative capacity and finally (v) inhibition of anti-tumor reactivity that is overcome by blocking NKG2A. From a clinical point of view, these results open a promising alternative for immunotherapies based on NKG2A blockade in CRC, which could be performed alone or in combination with other IC inhibitors, adoptive cell transfer or therapeutic vaccination.

Identification of TCR repertoires in functionally competent cytotoxic T cells cross-reactive to SARS-CoV-2

SARS-CoV-2-specific CD8+ T cells are scarce but detectable in unexposed healthy donors (UHDs). It remains unclear whether pre-existing human coronavirus (HCoV)-specific CD8+ T cells are converted to functionally competent T cells cross-reactive to SARS-CoV-2. Here, we identified the HLA-A24-high binding, immunodominant epitopes in SARS-CoV-2 spike region that can be recognized by seasonal coronavirus-specific CD8+ T cells from HLA-A24+ UHDs. Cross-reactive CD8+ T cells were clearly reduced in patients with hematological malignancy, who are usually immunosuppressed, compared to those in UHDs. Furthermore, we showed that CD8+ T cells in response to a selected dominant epitope display multifunctionality and cross-functionality across HCoVs in HLA-A24+ donors. Cross-reactivity of T-cell receptors isolated from them exhibited selective diversity at the single-cell level. Taken together, when stimulated well by immunodominant epitopes, selective pre-existing CD8+ T cells with high functional avidity may be cross-reactive against SARS-CoV-2.

An immunodominant NP105\u2013113-B*07:02 cytotoxic T cell response controls viral replication and is associated with less severe COVID-19 disease

NP105–113-B*07:02-specific CD8+ T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP105–113-B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with T cell receptor usage, transcriptome signature and disease severity (acute n = 77, convalescent n = 52). We demonstrated a beneficial association of NP105–113-B*07:02-specific T cells in COVID-19 disease progression, linked with expansion of T cell precursors, high functional avidity and antiviral effector function. Broad immune memory pools were narrowed postinfection but NP105–113-B*07:02-specific T cells were maintained 6 months after infection with preserved antiviral efficacy to the SARS-CoV-2 Victoria strain, as well as Alpha, Beta, Gamma and Delta variants. Our data show that NP105–113-B*07:02-specific T cell responses associate with mild disease and high antiviral efficacy, pointing to inclusion for future vaccine design.

Characterization of a library of 20 HBV-specific MHC class II-restricted T cell receptors

CD4+ T cells play an important role in the immune response against cancer and infectious diseases. However, mechanistic details of their helper function in hepatitis B virus (HBV) infection in particular, or their advantage for adoptive T cell therapy remain poorly understood as experimental and therapeutic tools are missing. Therefore, we identified, cloned, and characterized a comprehensive library of 20 MHC class II-restricted HBV-specific T cell receptors (TCRs) from donors with acute or resolved HBV infection. The TCRs were restricted by nine different MHC II molecules and specific for eight different epitopes derived from intracellularly processed HBV envelope, core, and polymerase proteins. Retroviral transduction resulted in a robust expression of all TCRs on primary T cells. A high functional avidity was measured for all TCRs specific for epitopes S17, S21, S36, and P774 (half-maximal effective concentration [EC50] <10 nM), or C61 and preS9 (EC50 <100 nM). Eight TCRs recognized peptide variants of HBV genotypes A to D. Both CD4+ and CD8+ T cells transduced with the MHC II-restricted TCRs were polyfunctional, producing interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and granzyme B (GrzB), and killed peptide-loaded target cells. Our set of MHC class II-restricted TCRs represents an important tool for elucidating CD4+ T cell help in viral infection with potential benefit for T cell therapy.

Identification of cross-reactive CD8+ T cell receptors with high functional avidity to a SARS-CoV-2 immunodominant epitope and its natural mutant variants

Despite the growing knowledge of T cell responses in COVID-19 patients, there is a lack of detailed characterizations for T cell-antigen interactions and T cell functions. Here, with a predicted peptide library from SARS-CoV-2 S and N proteins, we found that specific CD8+ T cell responses were identified in over 75% of COVID-19 convalescent patients (15/20) and an epitope from the N protein, N361-369 (KTFPPTEPK), was the most dominant epitope from our selected peptide library. Importantly, we discovered 2 N361-369-specific T cell receptors (TCRs) with high functional avidity that were independent of the CD8 co-receptor. These TCRs exhibited complementary cross-reactivity to several presently reported N361-369 mutant variants, as to the wild-type epitope. Further, the natural functions of these TCRs in the cytotoxic immunity against SARS-CoV-2 were determined with dendritic cells (DCs) and the lung organoid model. We found that the N361-369 epitope could be normally processed and endogenously presented by these different types of antigen presenting cells, to elicit successful activation and effective cytotoxicity of CD8+ T cells ex vivo. Our study evidenced potential mechanisms of cellular immunity to SARS-CoV-2, and illuminated potential ways of viral clearance in COVID-19 patients. These results indicate that utilizing CD8-independent TCRs against SARS-CoV-2-associated antigens may provide functional superiority that is beneficial for the adoptive cell immunotherapies based on natural or genetically engineered T cells. Additionally, this information is highly relevant for the development of the next-generation vaccines with protections against continuously emerged SARS-CoV-2 mutant strains.

A Shared SF3B1 Neoantigen Is Presented on Primary Malignant Cells and Induced Pluripotent Stem Cell-Derived Hematopoietic Lines

Myeloid malignancies are susceptible to immunologic destruction but antigen-specific T cell immunotherapies for these diseases are currently limited. Immunotherapies targeting neoantigens created from recurrent protein-coding mutations should selectively eradicate malignant cells and spare their normal counterparts to limit the risk of myeloablation. In particular, the paucity of neoplastic cells in MDS specimens impedes the identification and validation of targetable neoantigens for the development of immunotherapies for this disorder. Induced pluripotent stem cells (iPSC), generated from primary patient cells and differentiated into hematopoietic lines, can recapitulate the genotype and phenotype of the original disease (Hsu et al., Blood 2019) and could provide a renewable source of MDS progenitor cells for the development of novel T cell immunotherapy. SF3B1 mutations serve as a neoplastic driver for myeloid neoplasms (Papaemmanuil et al., Blood 2013), occuring in 20-30% of all MDS and &amp;gt;60% of MDS with ringed sideroblasts (Cazzola et al., Blood 2013). We hypothesized that SF3B1 mutations produce HLA-presented neoantigens, which can be targeted to eliminate primary MDS cells and iPSC with SF3B1 mutations. We interrogated the amino acid sequences from protein-coding mutations in SF3B1 using HLA-binding prediction algorithms (Immune Epitope Database Stabilized Matrix Method and Artificial Neural Network, netMHCpan 4.0) to predict binding to 20 prevalent HLA-A, -B, and -C alleles, and identified a candidate SF3B1mut epitope with strong predicted binding to HLA-B*40:01. HLA binding is necessary but not sufficient for T cell recognition of an epitope. We thus next evaluated the immunogenicity of the candidate SF3B1mut neoantigen epitope by stimulating CD8+ T cells isolated from healthy volunteer donors with peptide epitope-loaded autologous monocyte-derived dendritic cells, and isolated SF3B1mut -specific T cell clones, confirming the candidate epitope's immunogenicity. Two clones showed high functional avidity for the epitope in cytotoxicity assays (Figure 1A). To determine whether the epitope was naturally processed and presented by primary malignant myeloid cells, high-avidity SF3B1mut-specific clones were co-cultured with hematopoietic cells from patients with active MDS or acute myeloid leukemia (AML), then assessed for antigen recognition in a CD107a degranulation assay. High-avidity clones showed increased degranulation in response to a primary SF3B1mut HLA-B*40:01+ sample compared to samples lacking either the mutation or restricting HLA (Figure 1B), indicating that the SF3B1mut epitope is naturally processed and presented. We then evaluated whether SF3B1mut-specific clones could recognize SF3B1mut iPSC-derived hematopoietic cells. We reprogrammed hematopoietic stem/progenitor cells (HSPC) from SF3B1mut MDS patient bone marrow and established two iPSC lines: SF3B1mut and an isogenic control with wild-type SF3B1. Multipotent hematopoietic progenitor lines (MPP-5F) were then generated from iPSC lines by doxycycline-dependent expression of five HSPC transcription factors (Hsu et al., Blood 2019). In a CD107a degranulation assay, high-avidity clones showed ~30% degranulation with SF3B1mut MPP-5F, compared to ~5% with the isogenic control (Figure 1C), indicating that the iPSC-derived MPP-5F line recapitulated presentation of the neoantigen. We present a novel neoantigen with promise as an immunotherapy target for MDS, and for other hematologic malignancies with SF3B1 mutations including secondary AML, MDS/myeloproliferative neoplasm overlap syndrome, and advanced chronic lymphocytic leukemia (Cazzola et al., Blood 2013). Future experiments will assess the efficacy of SF3B1mut-specific T cell immunotherapy in vivo in a murine model and evaluate transfer of SF3B1mut-specific T cell receptors as a potential therapeutic approach. Our studies also indicate that iPSC-derived MPP-5F lines have potential to substitute for primary patient cells in neoantigen validation studies, a finding with broad utility for immunotherapy development in hematologic malignancies. Ongoing studies will assess MPP-5F presentation of a diverse panel of antigens, including minor histocompatibility antigens, non-mutated cancer antigens, and other neoantigens. Disclosures Oehler: Pfizer, Inc: Research Funding; Takeda: Consultancy; BMS: Consultancy. Bleakley:HighPass Bio: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties, Research Funding.

Fine tuning CD8+ T cell functional avidity by the PD-1/SHP2 axis

Abstract T cells of high functional avidity recognize low antigen levels and have the most effective cytotoxic responses against viruses and tumor cells. Understanding the molecular regulations of T cell functional avidity is essential to aid novel vaccine design as well as adoptive immunotherapy by improving the T cell quality. Programmed cell death protein 1 (PD-1) is known to negatively regulate the signal strength of T cell receptor (TCR). Activated PD-1 recruits Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (Shp2), which dephosphorylates kinases involved in TCR signaling. However, it is unclear whether PD-1/SHP2 can modulate T cell functional avidity. To address this question, we treated splenocytes from OT-1 transgenic mice with anti-PD-1 or SHP099 (SHP2 inhibitor), or a combination of both for 3 days in a selective culture condition of APC-pulsed stimulation with 10−6 M or 10−9 M SINNFEKL, respectively. Our preliminary data showed that blocking SHP2/PD-1 enhanced functional avidity and antigen sensitivity of CD8+ T cell in vitro when stimulated with limited SINNFEKL antigen. Blocking of PD1/SHP2 also enhanced CD8 beta expression, which has been shown correlated with high functional avidity CD8+ T cells. These data suggest that PD1/SHP2 is involved in regulating T cell functional avidity.

Massively parallel interrogation and mining of natively paired human TCRαβ repertoires.

T cells engineered to express antigen-specific T cell receptors (TCRs) are potent therapies for viral infections and cancer. However, efficient identification of clinical candidate TCRs is complicated by the size and complexity of T cell repertoires and the challenges of working with primary T cells. Here, we present a high-throughput method to identify TCRs with high functional avidity from diverse human T cell repertoires. The approach uses massively parallel microfluidics to generate libraries of natively paired, full-length TCRαβ clones, from millions of primary T cells, which are then expressed in Jurkat cells. The TCRαβ-Jurkat libraries enable repeated screening and panning for antigen-reactive TCRs using peptide:MHC binding and cellular activation. We captured >2.9 million natively paired TCRαβ clonotypes from six healthy human donors and identified rare (<0.001% frequency) viral antigen–reactive TCRs. We also mined a tumor-infiltrating lymphocyte (TIL) sample from a melanoma patient and identified several tumor-specific TCRs, which, after expression in primary T cells, led to tumor cell killing.

Effective Targeting of PRAME-Positive Tumors with Bispecific T Cell-Engaging Receptor (TCER®) Molecules

T cell receptors (TCRs) naturally recognize human leukocyte antigen (HLA)-bound peptides derived from foreign and endogenous proteins regardless of their extracellular or intracellular location. Preferentially expressed antigen in melanoma (PRAME) has been shown to be expressed at high levels in a variety of cancer cells while being absent or present only at very low levels in normal adult tissues except testis. In contrast to most other cancer/testis antigens, PRAME is expressed not only in solid tumors but also in leukemia and myeloma cells. Immunotherapy with bispecific T cell engagers has emerged as a novel and promising treatment modality for malignant diseases, however, antibody-based approaches (ie. blinatumomab) are restricted to few surface antigens such as CD19 or BCMA. Immatics has developed bispecific T cell-engaging receptors (TCER®) that are fusion proteins consisting of an affinity-maturated TCR and a humanized T cell-recruiting antibody with an effector function-silenced IgG1 Fc part. TCER® molecules confer extended half-life together with antibody-like stability and manufacturability characteristics. The molecular design allows for effective redirection of T cells towards target peptide-HLA selectively expressed in tumor tissues. Here we present proof-of-concept data from a TCER® program targeting a PRAME-derived peptide bound to HLA-A*02:01. We confirmed the abundant presence of the target peptide-HLA in several cancer indications and its absence in relevant human normal tissues by using the XPRESIDENT® target discovery engine, which combines quantitative mass spectrometry, transcriptomics and bioinformatics. Yeast surface display technology was used to maturate the stability and affinity of a parental human TCR recognizing PRAME with high functional avidity and specificity. During maturation we applied XPRESIDENT®-guided off-target toxicity screening, incorporating the world's largest normal tissue immunopeptidome database, to deselect cross-reactive candidate TCRs. The maturated TCRs were engineered into the TCER® scaffold and production in Chinese hamster ovary (CHO) cells generated highly stable molecules with low tendency for aggregation as confirmed during stress studies. Following TCR maturation, the TCER® molecules exhibited an up to 10,000-fold increased binding affinity towards PRAME when compared to the parental TCR. The high affinity correlated with potent in vitro anti-tumor activity requiring only low picomolar concentrations of TCER® molecules to induce half-maximal lysis of tumor cells expressing the target at physiological levels. Furthermore, using a tumor xenograft model in immunodeficient NOG mice, we could demonstrate significant growth inhibition of established tumors upon intravenous injection of TCER® molecules. Pharmacokinetic profiling in NOG mice determined a terminal half-life of more than 4 days, compatible with a once weekly dosing regimen in patients. For the safety assessment, we measured killing of more than 20 different human normal tissue cell types derived from high risk organs. Notably, we could confirm a favorable safety window for selected TCER® molecules, which induced killing of most normal tissue cells only at significantly higher concentrations than required for killing of tumor cells. To further support safety of TCER® molecules, we also performed a comprehensive characterization of potential off-target peptides selected from the XPRESIDENT® normal tissue database based on its high similarity to the sequence of the target peptide or based on data from alternative screening approaches. In summary, the efficacy, safety and manufacturability data to be presented provide preclinical proof-of-concept for a novel bispecific T cell-engaging receptor (TCER®) molecule targeting PRAME for treatment of various malignant diseases. Disclosures Bunk: Immatics: Employment. Hofmann:Immatics: Employment. Unverdorben:Immatics: Employment. Hutt:Immatics: Employment. Pszolla:Immatics: Employment. Schwöbel:Immatics: Employment. Wagner:Immatics: Employment. Yousef:Immatics: Employment. Schuster:Immatics: Employment. Missel:Immatics: Employment. Schoor:Immatics: Employment. Weinschenk:Immatics: Employment, Equity Ownership. Singh-Jasuja:Immatics: Employment, Equity Ownership. Maurer:Immatics: Employment. Reinhardt:Immatics: Employment, Equity Ownership.

FT576: A Novel Multiplexed Engineered Off-the-Shelf Natural Killer Cell Immunotherapy for the Dual-Targeting of CD38 and Bcma for the Treatment of Multiple Myeloma

Multiple myeloma (MM) is a B cell neoplasm that originates from the malignant transformation of plasma cells, with treatment strategies that include chemotherapeutic agents and immunomodulatory drugs. Recently, significant effort has been applied to the development of monoclonal antibody (mAb) and chimeric antigen receptor (CAR) T cell therapies for the treatment of advanced MM. Anti-CD38 mAb therapy is at the forefront of these efforts, with clearly demonstrated clinical benefit and availability of a FDA-approved mAb in daratumumab. Antibody-dependent cellular cytotoxicity (ADCC) is a key mechanism of action of CD38-targeted mAbs; however, high CD38 expression on natural killer (NK) cells results in fratricide, which depletes the NK cells necessary for ADCC. In addition to CD38, targeting of other MM-associated cell-surface proteins has been explored. Of these antigens, the TNF-superfamily member BCMA is among the most researched and is under development by multiple groups as a CAR target. Several clinical trials in MM have shown promising initial results targeting BCMA with CAR T cells, however there remains significant opportunity to improve both relapse rates and treatment of relapsed patients. Collectively, clinical data would suggest that combinatorial targeting of both CD38 and BCMA may improve clinical efficacy compared with targeting either antigen alone. We have developed a multiple-target, adoptive NK cell immunotherapy approach for the treatment of MM. The strategy utilizes our off-the-shelf NK cell platform with four engineered attributes: 1) an anti-BCMA CAR for direct MM targeting, 2) high affinity non-cleavable CD16 (hnCD16) for enhanced ADCC in combination with anti-CD38 mAbs, 3) CD38 deletion for resistance to anti-CD38 mAb induced NK cell depletion, and 4) IL-15/IL-15 receptor α fusion protein (IL-15RF; IL-15 fused to IL-15Rα) for enhanced NK cell persistence. The anti-BCMA CAR consists of a unique single chain variable fragment (scFv) targeting domain with a BCMA binding affinity in the low nanomolar range, providing high functional avidity and efficacy in disease settings where BCMA antigen density is low. Our approach utilizes NK cells derived from a genetically engineered, clonally-derived master pluripotent stem cell line with uniform expression of anti-BCMA CAR, IL-15RF, hnCD16, and CD38 bi-allelic knockout. The engineered master pluripotent stem cell line serves as the starting material for consistent and repeatable manufacture of off-the-shelf NK cells that contain all described attributes in a homogenous manner (termed FT576) and that can be produced at a scale to support multi-dose treatment strategies and on-demand dose availability. In preclinical studies, FT576 NK cells exhibited uniform expression of CD16, CAR, and IL15-RF and did not express CD38 (&lt;1%). In an in vitro fratricide assay, FT576 NK cells were entirely resistant to daratumumab-induced fratricide, with no detectable specific cytotoxicity when exposed to increasing concentrations of daratumumab. Conversely, peripheral blood NK cells were sensitive to daratumumab-induced fratricide (up to 33% cytotoxicity within 3 hrs of daratumumab exposure). FT576 NK cells demonstrated enhanced cytotoxicity against the MM1.S MM cell line during a long-term cytotoxicity assay compared with control NK cells that lacked CAR expression (62% cytotoxicity for FT576 vs 26% for control). In addition, cellular persistence was greater than NK cells lacking the IL-15RF protein, and FT576 NK cells demonstrated the unique ability to expand in vitro absent of exogenous cytokine support (61-fold expansion vs. 4-fold for IL-15RF negative). Importantly, FT576 NK cells remained ADCC competent, as combination with daratumumab enhanced cytotoxicity against MM cell lines in a 2D cytotoxicity assay. Additionally, FT576 mediated direct cytotoxicity against RPMI-8226 MM spheroids, leading to &gt;99% cytotoxicity in a 3D-spheroid culture model. Preclinical studies are ongoing to support the advancement of FT576 as the first-of-kind cellular therapeutic for the combination of anti-BCMA CAR and mAb-directed targeting of MM. Disclosures Bjordahl: Fate Therapeutics, Inc.: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Goodridge:FATE THERAPEUTICS: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Robinson:Fate Therapeutics, Inc.: Employment. Ruller:Fate Therapeutics, Inc.: Employment. Pribadi:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Abujarour:Fate Therapeutics, Inc.: Employment. Dinella:Fate Therapeutics, Inc.: Employment. Huffman:Fate Therapeutics, Inc.: Employment. Chu:FATE THERAPEUTICS: Employment. Valamehr:Fate Therapeutics, Inc: Employment.

Abstract B118: Development of a novel prostatic acid phosphatase-derived vaccine for the treatment of advanced prostate cancer

Abstract Our aim is to develop a new and more effective prostatic acid phosphatase (PAP) peptide-based vaccine for the treatment of advanced prostate cancer (PCa). We have previously shown that a 15mer PAP peptide-derived vaccine, when administered as a DNA vaccine, could induce PAP-specific T-cell responses and reduce the growth of implanted murine TRAMP C1-derived tumors in a syngeneic heterotopic murine model. We have subsequently developed an elongated (42mer) PAP-derived peptide in which Alanine at position 115 was replaced by Leucine.Herein, the capacity of the wild-type and mutated PAP-42mer vaccines to induce PAP-specific immune responses were compared in two pre-clinical murine models. The murine PAP-42mer sequence was evaluated in C57Bl/6 mice and the human PAP-42mer sequence in HHDII/DR1 transgenic mice. Following the identification of the most promising PAP-42mer sequence, the capacities of different delivery systems (CpG adjuvant, CAF09 adjuvant and ImmunoBody® DNA vaccine) to generate PAP-specific immunity were compared. For these studies, the responsiveness of splenocytes isolated from immunized mice following in vitro stimulation with MHC class I or class II wild-type vaccine-derived peptides (8/9 or 15 amino-acid long) was assessed using an IFNγ ELISpot assay and by immunophenotyping of splenic T-cells using flow cytometry. The ability of splenocytes to kill relevant targeT-cells after a short in vitro stimulation was assessed using a 51chromium release assay. B16 cells that had been knocked out for beta-2m gene and transfected to express chimeric HLA-A2 (HHDII), HLA-DR1 and human PAP implanted into HHDII/DR1 mice was used as a proof-of-concept model to assess the antitumor efficacy of the hPAP42mer mutated vaccine with CAF09 adjuvant in a prophylactic setting.The mutated murine and human PAP42mer-based vaccines induced a higher number of IFNγ-releasing splenocytes in response to in vitro stimulation with class I or class II vaccine-derived peptides and generated cells having a higher functional avidity. CAF09 and ImmunoBody® were superior to CpG in inducing PAP-specific immune responses, with CAF09 eliciting strong immune responses in both models, and ImmunoBody® eliciting potent immune responses in C57Bl/6 mice. Indeed, a higher proportion of CD8+ T-cells were able to release IFNγ and TNFα, to proliferate (Ki67 expression) and to degranulate (CD107a and Granzyme B expression) after short incubation with MHC class-I peptide. These vaccines strategies were also able to induce a higher proportion of memory CD8+ T-cells and the expression of PD-1 on CD8+ T-cells. Additionally, splenocytes from vaccinated mice were able to kill MHC class I peptide-pulsed targeT-cells (T2 and RMAS cells) and murine PCa cells expressing PAP (TRAMP-C1 cells) in vitro. The vaccine slowed the growth of human-PAP+-B16-derived tumors in HHDII/DR1 model and prolonged the survival (9 days delay for 30% of the mice). Splenic CD8+ T-cells and CD8+ tumor-infiltrating cells from vaccinated mice released IFNγ and TNFα, proliferated and degranulated in response to in vitro stimulation with MHC class I peptide. Finally, multimer-based flow cytometric analysis of peripheral blood mononuclear cells from patients with PCa following in vitro stimulation with PAP-derived peptides demonstrated the presence of PAP42mer-reactive CD8+ T-cells. Ongoing studies are assessing the effect of combining the vaccine with checkpoint inhibition (a PD-1 monoclonal antibody) in a therapeutic setting. In summary, we have developed a vaccine strategy which induces robust anti-PAP immunity and slows the growth of implanted PAP+ cancer cell-derived tumors in vivo. The presence of PAP-specific CD8+ T-cells in the periphery of patients with advanced PCa suggests that these patients will benefit from this new approach, especially when combined with checkpoint inhibition. Citation Format: Pauline Le Vu, Jayakumar Vadakekolathu, Dennis Christensen, Lindy Durrant, Graham Pockley, Stephanie E.B. McArdle. Development of a novel prostatic acid phosphatase-derived vaccine for the treatment of advanced prostate cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B118.

CAR T Cells with Enhanced Sensitivity to B Cell Maturation Antigen for the Targeting of B Cell Non-Hodgkin's Lymphoma and Multiple Myeloma.

Autologous Tcells genetically modified with a chimeric antigen receptor (CAR) redirected at CD19 have potent activity in the treatment of B cell leukemia and B cell non-Hodgkin's lymphoma (B-NHL). Immunotherapies to treat multiple myeloma (MM) targeted the B cell maturation antigen (BCMA), which is expressed in most cases of MM. We developed a humanized CAR with specificity for BCMA based on our previously generated anti-BCMA monoclonal antibody. The targeting single-chain variable fragment (scFv) domain exhibited a binding affinity in the low nanomolar range, conferring Tcells with high functional avidity. Redirecting Tcells by this CAR allowed us to explore BCMA as an alternative target for mature B-NHLs. We validated BCMA expression in diffuse large Bcell lymphoma, follicular lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia. BCMA CAR Tcells triggered target cell lysis with an activation threshold in the range of 100 BCMA molecules, which allowed for an efficient eradication of B-NHL cells invitro and invivo. Our data corroborate BCMA is a suitable target in B cell tumors beyond MM, providing a novel therapeutic option for patients where BCMA is expressed at low abundance or where anti-CD19 immunotherapies have failed due to antigen loss.

PS-031 - T cells grafted with HBV-specific T-cell receptors of high functional avidity achieve functional cure of HBV infection in humanized mice

PS-031 - T cells grafted with HBV-specific T-cell receptors of high functional avidity achieve functional cure of HBV infection in humanized mice