Endogenous TCRs Research Articles

Abstract 26: An HLA-class II restricted HPV18 E7 specific TCR cloned from a long-term surviving cervical cancer patient

Abstract T cell receptor (TCR)-engineered T cell therapy is a promising approach for the treatment of solid malignancy, with multiple clinical trials reporting impressive responses using affinity-matured TCRs. However, artificially mutated TCRs can increase the risk of off-target toxicities due to unexpected cross-reactions, which limits the overall safety of this approach. To tackle this challenge, we have developed a novel strategy for cloning tumor-specific endogenous therapeutic TCRs from long-term surviving patients who have responded to immunotherapy. Specifically, we have identified a TCR (10F04) that is specific to human papillomavirus type 18 (HPV18) E784-98 and restricted to human leukocyte antigen (HLA) class II molecular DRA/DRB1*09:01 from the peripheral blood of a patient with HPV-positive metastatic cervical cancer who received multiple antigens stimulating cellular therapy (MASCT) and exhibited sustained T-cell immunological responses. Upon transduction into human T cells, the 10F04 TCR demonstrated robust antitumor activity in both in vitro and in vivo models. Notably, the TCR effectively redirected both CD4 and CD8 T cells to specifically recognize tumor cells and induced multiple cytokine secretion along with durable antitumor activity. Importantly, no cross-reactivity was detected in toxicity studies. As a result, this TCR is currently being investigated in a phase I clinical trial for treating HPV18-positive cancers (NCT05787535). The first-in-human infusion had been just completed. No severe adverse event had been observed, while an early proliferation of TCR-T was detected several days after infusion. Our approach of cloning tumor-specific and safe endogenous therapeutic TCRs from long-term surviving patients offers a promising strategy for developing safe and effective TCR-engineered T-cell therapies for solid tumors. These findings provide important insights into the potential of HLA class II-restricted TCR-T therapy as a novel cancer treatment modality. Citation Format: Jianting Long, Xihe Chen, Shudan Ou, Minjun Ma, Junjun Chu, Yanyan Han. An HLA-class II restricted HPV18 E7 specific TCR cloned from a long-term surviving cervical cancer patient [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 26.

Abstract 6717: Targeting mutant KRAS proteins with novel TCR-mimic fully human antibodies

Abstract Mutated KRAS proteins are ideal cancer targets, as they are expressed frequently and specifically in certain solid tumors. A large proportion of human colorectal cancer and pancreatic ductal adenocarcinoma express the tumor driver KRAS gene mutations G12V/G12D, but drugs targeting G12V/G12D are not available, revealing a huge unmet clinical need. While small molecules often fail to target the KRAS mutation G12V/G12D, T cell receptor-mimic (TCR-mimic) antibodies can specifically recognize KRAS mutations presented by human leukocyte antigen (HLA), opening up possibilities for targeting such intracellular antigens. Here, we discovered novel antibodies highly specific to G12V/HLA and G12D/HLA complexes by immunizing our proprietary RenTCR-mimicTM mice and using high-throughput Beacon-based screening. These TCR-mimic antibodies have higher affinities compared to endogenous TCRs, which may effectively reduce the possibility of tumor escape. Germline distribution analysis indicated their high sequence diversity, which suggests diverse epitope targeting. Although pancreatic cancer is extremely difficult to treat and has an extremely low KRAS mutant peptide-HLA complex density on the cell surface, our TCR-mimic antibodies exhibited potent in vitro tumor lysis activity when assembled into CD3 T cell engagers. Furthermore, these antibodies demonstrated convincing off-target safety. Together, our results indicate promising therapeutic potential of these KRAS mutation-targeted TCR-mimic antibodies for the treatment of solid tumors. Citation Format: Jun Du, Wanbo Tang, Xin Jiao, Limin Zhao, Pengfei Du, Yuqi Zhang, Jian Bao, Han Chen, Chaoshe Guo, W. Frank An. Targeting mutant KRAS proteins with novel TCR-mimic fully human antibodies [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 6717.

Enhancing MHC-Independent T Cell Receptors Using Rational Protein Engineering

The paradigm of synthetic receptors, chimeric antigen receptors (CARs), combine MHC-independence with T cell activation. While T cells engineered to express CARs are successful for some patients, the majority are not cured and clinical failures often result from CAR-driven T cell dysfunction (Lynn R, Nature 2019; Selli ME, Blood 2023). Simultaneously, many patients develop cytokine release syndrome, a hyperinflammatory state resulting from uncontrolled CAR-driven immune activation, indicating that CARs can drive both failure and toxicity. We hypothesized that these limitations result from CAR design which, in its simplicity, bypasses natural TCR regulation. To re-engage endogenous T cell regulatory circuits, we replaced the variable region of the TCR α and β chains with the CD19 binding domain used in several clinical CAR constructs. As shown recently by others (Liu Y, Sci Transl Med 2021; Mansilla-Soto Nature Medicine 2022), we found that this format of synthetic receptor expressed on the cell surface, clustered with endogenous CD3 molecules, and drove T cell cytotoxicity against CD19+ leukemia (Nalm6). Intriguingly, surface expression of these MHC-independent TCRs (miTCR) was markedly reduced in comparison to transgenic full-length TCRs and CARs. Examination of the chimeric interface created by joining the antibody and TCR parent chains revealed significant biochemical conflicts that, based on predictive modeling, disrupted naturally-occurring hydrostatic interactions and associated hydrogen bonds, collectively resulting in steric “strain” on the miTCR. We generated a series of miTCR mutants which resolved these conflicts in a step-wise fashion to more closely approximate the “relaxed” state of endogenous TCRs. We found that most mutations improved miTCR surface expression, and more importantly that some mutations significantly boosted T cell functionality as compared to the wild-type miTCR. Impressively, one mutant that closely approximated endogenous TCR structure (mut035) enabled functionality similar to CD19 CARs in short-term cytotoxicity assays. To further evaluate the functional implications of targeted interface mutation, we subjected miTCRs to chronic in vitro stimulation assays in which engineered T cells are exposed to high quantities of Nalm6 for prolonged periods until T cell lose function. We observed that mut035 outperformed all other miTCRs, but all miTCR T cells lost the ability to kill targets earlier than CAR T cells. To determine if a lack of costimulation was the cause of this reduced efficacy we engineered Nalm6 cells to express ligands for CD28 (CD80) and 41BB (41BBL). In the presence of these ligands, long-term functionality all miTCR T cells was profoundly improved. Notably, mut035 T cells persisted longer and killed Nalm6 better than CD19 CAR T cells under conditions of persistent and high-intensity stimulation. Finally, to determine if CD28 or 41BB engagement was most supportive of miTCR function we designed a “priming” experiment. miTCR T cells were initially cultured with Nalm6 expressing either no, one or both costimulatory ligands and then, after tumor clearance, were re-challenged with WT Nalm6, mirroring endogenous immune responses to infection. mut035 T cells initially exposed to CD80 were unable to clear WT cells on re-challenge, however priming with 41BBL enabled potent and prolonged co-stimulation-independent killing of large Nalm6 burdens, suggesting engagement of endogenous T cell functionality. These data demonstrate that classical approaches to combine protein domains can lead to biochemical conflicts that restrain chimeric protein function. Targeted mutation significantly enhanced miTCR function to exceed CAR under conditions of high-burden, high-antigen tumors.

Harnessing the Full Potential of Regulatory T Cells by mRNA-induced Antigen Specificity

Abstract Regulatory T cells (Tregs) are crucial in inducing and maintaining tolerance, and already have shown clinical potential. In this study, we describe an efficient non-viral engineering method by TCR-encoding mRNA electroporation, while also exploring the potential of further promoting on-target effects by disrupting the endogenous TCR using a fully RNA-based CRISPR/Cas9 technology. Endogenous TCR disruption prior to TCR-transfection resulted in more than 90% blockade of the endogenous TCR expression and a similar high (>85%) but more homogenous expression of the transgenic TCR. Importantly, following antigen-specific TCR-induced stimulation, both engineered Tregs significantly increased the expression of multiple activation and suppression markers. Moreover, the engineered Tregs outcompeted polyclonal Tregs in suppressing antigen-specific effector T cell proliferation and performed better in blocking moDC maturation, even in the presence of antigen-activated effector T cells. Secretome screening revealed a distinct anti-inflammatory profile for TCR-Tregs. On the other hand, TCR-engineered KO Tregs co-expressed anti-inflammatory cytokines together with some pro-inflammatory cytokines, but qualitatively different from their effector Th counterparts. Hence, an anti-inflammatory profile with Th cell features is seen with TCR-engineered KO Tregs. In conclusion, we show that this versatile approach can be used to induce the functional expression of a transgenic TCR, but also for CRISPR/Cas9-mediated disruption of endogenous TCRs with high efficiency using an RNA-based platform. Comparison between polyclonal Tregs and TCR engineered Tregs in EAE using an HLA-transgenic mouse model is now being investigated. National Multiple Sclerosis Society (RG-1612-26484)

Gene modified NK cell line as a powerful tool for evaluation of cloned TCRs for TCR-T cell therapy

T cell receptor-engineered T cell (TCR-T) therapy is anticipated as a next generation-immunotherapy for cancer and recent advances of TCR isolation technology have enabled patient’s T cells to express TCRs recognizing multiple combinations of specific peptides and human leukocyte antigens (HLA). However, evaluation processes for the TCR-induced cytotoxicity activity using primary T cells are laborious and time-consuming. In this study, we established a cell line that do not express endogenous TCRs, enabling to generate large numbers of homogeneous cells, and can measure the cytotoxic activity of the isolated TCRs. To this end, we transduced a Natural Killer (NK) cell line with human CD3 molecules and interleukin (IL)-2. The TCR expressing NK cells killed target cells as similarly to TCR-transduced primary T cells and secreted various cytokines/chemokines including IL-2. Thus, the gene-modified NK cell can be a powerful tool to rapidly and efficiently evaluate the functions of isolated TCRs.

CRISPR-Mediated Base Conversion Allows Discriminatory Depletion of Endogenous T Cell Receptors for Enhanced Synthetic Immunity

Emerging base editing technology exploits CRISPR RNA-guided DNA modification effects for highly specific C > T conversion, which has been used to efficiently disrupt gene expression. These tools can enhance synthetic T cell immunity by restricting specificity, addressing histocompatibility leukocyte antigen (HLA) barriers, and promoting persistence. We report lentiviral delivery of a hepatitis B-virus (HBV)-specific recombinant T cell receptor (rTCR) and a linked CRISPR single-guide RNA for simultaneous disruption of endogenous TCRs (eTCRs) when combined with transient cytosine deamination. Discriminatory depletion of eTCR and coupled expression of rTCR resulted in enrichment of HBV-specific populations from 55% (SEM, ±2.4%) to 95% (SEM, ±0.5%). Intensity of rTCR expression increased 1.8- to 2.9-fold compared to that in cells retaining their competing eTCR, and increased cytokine production and killing of HBV antigen-expressing hepatoma cells in a 3D microfluidic model were exhibited. Molecular signatures confirmed that seamless conversion of C > T (G > A) had created a premature stop codon in TCR beta constant 1/2 loci, with no notable activity at predicted off-target sites. Thus, targeted disruption of eTCR by cytosine deamination and discriminatory enrichment of antigen-specific T cells offers the prospect of enhanced, more specific T cell therapies against HBV-associated hepatocellular carcinoma (HCC) as well as other viral and tumor antigens.

Abstract 2310: Functional demonstration of CD19 chimeric antigen receptor (CAR) engineered Epstein-Barr virus (EBV) specific T cells: An off-the-shelf, allogeneic CAR T-cell immunotherapy platform

Abstract Chimeric antigen receptor (CAR) T cells have transformed the treatment of advanced B cell malignancies, but broad application has been limited due to technical and operational challenges in the autologous approach. Virus-specific T cells maintain native T cell receptors and have an established clinical profile and potential utility as an off-the-shelf, allogeneic CAR T-cell immunotherapy platform. Tabelecleucel (tab-cel®) is an investigational off-the-shelf, allogeneic T-cell immunotherapy that utilizes endogenous T cell receptors targeting EBV antigens associated with select lymphomas and solid tumors. Tab-cel® functions through endogenous TCRs restricted to EBV antigens and has been shown to be generally well tolerated with low incidence of GvHD and cytokine release syndrome, as well as efficacy in EBV+ post-transplant lymphoproliferative disorders (PTLD). Introduction of CAR transgenes into EBV-specific T cells provides an appealing approach for developing off-the-shelf, allogeneic CAR T immunotherapies. To evaluate feasibility for this platform, we engineered EBV-specific T cells to express second-generation CD19 CARs, utilizing CD28 or 4-1BB co-stimulatory domains. Resulting allo-EBV.CD19.CAR T cells exhibit high expression of both the CD19 CAR and EBV TCR. Additionally, allo-EBV.CD19.CAR T cells demonstrate an enriched central memory phenotype with higher frequency expression of CD62L, CCR7, and CD45RO. Allo-EBV.CD19.CAR T cells also show increased frequency of activation markers CD25 and 4-1BB, and produce activation-associated levels of IFN-γ comparable to EBV-specific T cells. Allo-EBV.CD19.CAR T cells exert potent and specific cytotoxicity against CD19-positive Nalm6 and Raji cells but have limited activity against CD19-negative K562 cells. Both CD19.CAR T and allo-EBV.CD19.CAR T-mediated cytotoxicity displayed rapid and comparable kinetics. Stimulation of allo-EBV.CD19.CAR T cells with either EBV- or CD19-positive targets also induced robust proliferation over several days of antigen challenge. Like unmodified EBV-specific T cells, allo-EBV.CD19.CAR T cells retain the ability to kill B-lymphoblastoid cell lines (BLCL) but spare autologous and allogeneic PHA-blast targets lacking CD19 and EBV antigen expression. This specificity in cytotoxicity is further supported by inflammatory cytokine profiles of effector-target cultures. From these results, allo-EBV.CD19.CAR T cells demonstrate efficient and effective lysis of antigen-positive target cells, while maintaining low allo-cytotoxic potential. These findings establish feasibility for engineering EBV-specific T cells by leveraging next-generation CAR technologies, and support further development as an off-the-shelf, allogeneic CAR-T immunotherapy platform to generate IND clinical candidates. Citation Format: Rhine R. Shen, Christina D. Pham, Michelle Min Wu, Daniel J. Munson, Blake T. Aftab. Functional demonstration of CD19 chimeric antigen receptor (CAR) engineered Epstein-Barr virus (EBV) specific T cells: An off-the-shelf, allogeneic CAR T-cell immunotherapy platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2310.

PF439 EXPLORING THE THERAPEUTIC POTENTIAL OF T‐CELL‐RECEPTOR REDIRECTED CD3+ NATURAL KILLER CELLS FOR ADOPTIVE CELLULAR THERAPY TO ACUTE MYELOID LEUKEMIA

Background:Adoptive cellular therapy (ACT) with redirected T cells expressing a chimeric antigen receptor (CAR) or transgenic T‐cell‐receptor (tTCR) has revolutionized cellular immunotherapy to hematological neoplasia, in particular to acute lymphoid leukemia, and also shows great promise as therapy for solid tumors. While CARs can only detect fully cell surface expressed target structures, TCR‐mediated recognition is not limited to surface antigens, but covers processed tumor neoantigens derived from the whole proteome. However, mispairing of transgenic and endogenous TCRs and restriction to patient‐derived, autologous T lymphocytes with variable “fitness” and T cell subsets due to individual health conditions and age of the patient exemplify the current limitations encountered in TCR‐redirected ACT. The natural killer (NK) cell line NK92 elicits lytic activity comparable to T cells, has been approved by the FDA for ACT and shown not to cause graft‐vs‐host disease. Since NK cell redirection is currently limited to CARs, NK92 cells engineered to express a CD3/TCR or CD3/CD8/TCR complex might evolve as an attractive, standardized cellular source for off‐the‐shelf TCR‐based ACT.Aims:Thus, the goal of this study was to explore NK92‐CD3+ and NK92‐CD3+CD8+ variants redirected to acute myeloid leukemia (AML) by expression of different AML‐reactive TCRs for antileukemic immunity in vitro and in vivo.Methods:NK92 cells expressing the γδεζ‐chains of the CD3 complex (NK92CD3+) were kindly provided by Dr. C. Wölfel (III. Dept. of Medicine) and further genetically engineered to express different human CD8 isoforms. Upon retroviral transfer (RT) of 3 different codon‐optimized and murinized TCRs obtained from AML‐specific CD8+CTL clones into NK92‐CD3+cells TCR expression was examined by flow cytometry (FC). NK92CD3+TCR+as well as NK92CD3+CD8+TCR+cells (together referred to as NK92TCR+) were enriched using antibiotic selection and immunomagnetic cell sorting (MACS®). Biological reactivity of redirected NK92TCR+cells to patient‐derived (primary) AML blasts was tested in vitro by IFN‐γ ELISPOT and 51Cr‐release assays. Comparable expression‐profiling of checkpoint molecules on NK92 and NK92TCR+cells such as e.g. CD80/86, PD1/PDL1, NKG2A, TIM3, TIGIT and HLA‐E was performed by FC. A patient‐derived AML‐xenograft (PDX) model in NSG mice has been already established.Results:Following RT and enrichment >80% of NK92‐CD3+cells expressed a given TCR, and >90% of NK92CD3+CD8+TCR+cells could be obtained. Upon coculture with different primary AML samples and corresponding EBV‐LCL blasts as controls NK92CD3+TCR+ elicited TCR dependent IFN–γ release and exhibited cytolytic activity to some but not all AML samples. CD8 coexpression slightly increased reactivity as measured by IFN‐γ release. Interestingly, as TCR‐redirected NK92 antileukemic responses appeared to be inhibited to some but not all AML samples we found high expression of HLA‐E on these AML blasts and of NGK2A on NK92TCR+ cells. Current blocking studies to examine whether a HLA‐E/NKG2A interaction might impair NK92TCR+ mediated AML reactivity are ongoing and will be presented. Moreover, first preliminary in vivo experiments of adoptively transferred NK92TCR+ cells into a NSG‐AML PDX model indicated biological activity but further studies are in progress to evaluate this point in more detail.Summary/Conclusion:These studies demonstrate that the established and FDA approved NK92 cell line can be redirected to elicit TCR‐mediated antitumoral immunity to AML. NK92TCR+ cells might thus represent a promising universal tool for an ‘off‐the‐shelf’ ACT product

Endogenous Secondary TCRs Promote Homeostatic Advantage Via Recognition of Self-Antigens

Abstract Our laboratory studies how non-thymically selected secondary TCRs contribute to the T cell repertoire. We have previously demonstrated that peripheral T cells naturally expressing 2 TCRs have increased response to autoantigens. We hypothesized that this could promote homeostatic proliferation of T cells expressing 2 TCRs, which may cause autoimmune risk. To examine secondary TCRs in homeostasis, we performed competitive transfers of CFSE-labeled T cells from B6.Ly5.1 and B6.TCRα+/−.Thy1.1 (lacking secondary TCRs) into irradiated B6 recipients. T cells injected at a 1:1 ratio demonstrated a disadvantage for TCRα+/− T cells in lymphopenia-induced proliferation (LIP), with TCRα+/−/wt donor cell ratios of 0.6±0.1 at 8 d after transfer and 0.5±0.4 at 28 d after transfer. T cell population skewing resulted from increased proliferation of wild-type cells. The homeostatic advantage of secondary TCRs was less in chronic LIP (cLIP) (transfer into TCRα-knockout mice), with TCRα+/−/wt donor cell ratios of 0.8±0.3 at 8 d after transfer and 0.7±0.3 at 28 d after transfer. There was no difference between wt and TCRα+/− T cells in proliferative response to stimulation via anti-CD3/CD28, nor were differences observed for expression of CD3, CD5, or CD127. LIP and cLIP depend on signaling through the TCR, though reactivity to self-antigens is thought to drive LIP as opposed to endogenous antigens from commensal microflora in cLIP. Our results suggest that the advantage for secondary TCRs in homeostatic expansion reflects increased ability to interact with self-pMHC antigens. Current effort is focused to understand how this affects protective and autoreactive peripheral T cell repertoires during aging or iatrogenic lymphopenia.

P3.02c-062 Anti-Lung Cancer Effect of CD8+ T Cells Transduced Retroviral Vector Encoding WT1-Specific TCRs and siRNAs Targeting Endogenous TCRs: Topic: IT Biomarkers

P3.02c-062 Anti-Lung Cancer Effect of CD8+ T Cells Transduced Retroviral Vector Encoding WT1-Specific TCRs and siRNAs Targeting Endogenous TCRs: Topic: IT Biomarkers

An optimized single chain TCR scaffold relying on the assembly with the native CD3-complex prevents residual mispairing with endogenous TCRs in human T-cells.

Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/β T-cell receptors (TCRα/β). However, potential mispairing of introduced TCRα/β-chains with endogenous β/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vβ-fragment to the TCR Cβ-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/β-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vβ-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vβ. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/β-positive T-cells.

Abstract A095: New genes for enhancing T cell function in adoptive cell therapy of cancer

Abstract A growing number of clinical trials point to the exquisite ability of adoptively transferred T cells to eradicate vast masses of tumor cells, including aggressive metastatic tumors. In one therapeutic approach T cells are derived from tumor biopsies (tumor-infiltrating lymphocytes, or TILs) and express endogenous TCRs. In others T cells are expanded from the peripheral blood lymphocyte pool and redirected to recognize tumor antigens by virtue of exogenous TCR or chimeric antigen receptor (CAR) genes. Although reports on enduring clinical responses are encouraging, a significant portion of patients who do not respond, or respond only partially and transiently, underscore the critical challenge of improving T cell function and survival at the tumor site. In attempt to improve the clinical efficacy of adoptive cell therapy we created three classes of genetic adjuvants designed to operate autonomously in gene-modified T cells. One class encodes constitutively-active (ca) toll-like receptors devoid of their ligand-binding domain, the other produces membrane-anchored immunostimulatory cytokines and the third encodes a novel homo-oligomerizing configuration of tumor necrosis factor receptors, inducing constitutive ligand-free activation. For gene delivery into human TILs or peripheral blood T cells we have been using electroporation of in-vitro-transcribed mRNA. Here we present data obtained in preliminary ex-vivo experiments designed to assess the function of caTLR4, membrane IL-2, IL-12 (single chain) and IL-15 and trimeric caCD40 and their level of cooperation. The mere expression of caTLR4 mRNA in polyclonal human CD8 and CD4 T cells induced the production of IFN-γ, triggered the surface expression of CD25, CD69 and 4-1BB and upregulated a panel of cytokines and chemokines. In all samples of anti-melanoma TILs tested, caTLR4 induced a burst of IFN-γ secretion, which usually ceased within 24 hours post-transfection. Furthermore, caTLR4 enhanced the cytolytic activity of TILs and augmented the secretion of IFN-γ, TNF-α and GM-CSF in the presence of autologous, but not mismatched, melanoma for at least 4 days post-transfection. The membrane cytokines were found to act mainly in-cis and each could support the proliferation of human CD8 and CD4 T cells for at least 6 days post-transfection, at a magnitude comparable to a saturating dose of soluble IL-2. Following mRNA electroporation of anti-melanoma TILs, membrane cytokines cooperated with caTLR4 in inducing IFN-γ secretion and upregulating the activation and costimulatory molecules CD25, CD69, 4-1BB and OX40. Different combinations of caTLR4, membrane cytokines and caCD40 mRNA induced spontaneous cytokine secretion in mRNA-transfected TILs, typically acting in a synergistic manner. Even when the spontaneous, initial effect waned, a considerably higher fraction of mRNA-transfected, compared to control-transfected TILs could still respond robustly to autologous, but not allogeneic, melanoma. In the presence of the three classes of adjuvants, transfected short-term cultured ‘young’ TILs revealed exceptional upregulation of 4-1BB, OX40, CD25 and CD28 and an increase in IFN-γ production and no, or only minimal change in the inhibitory receptors PD-1 and CTLA-4. Here, too, the different adjuvants displayed marked synergy, with caCD40 often exerting a prominent effect even when delivered as a single gene. Enhanced killing of autologous melanoma cells by electroporated young TILs was observed up to ten days post-electroporation. Our findings suggest that this new panel of genetic adjuvants can substantially improve the functional properties of antitumor T cells for extended periods, offering a new tool in all approaches for cancer immunotherapy employing adoptive T cell transfer. Citation Format: Gideon Gross, Aviad Pato, Hadas Weinstein-Marom, Noam Levin, Alon Margalit, Orit Itzhaki, Michal Besser, Tamar Peretz, Arthur Machlenkin, Michal Lotem. New genes for enhancing T cell function in adoptive cell therapy of cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A095.

16. Effective and Safe Gene-Modified T Cells Expressing High Affinity NYESO-1 Specific TCR Variants with Silencing Endogenous TCRs

16. Effective and Safe Gene-Modified T Cells Expressing High Affinity NYESO-1 Specific TCR Variants with Silencing Endogenous TCRs

Augmented Expression of WT1-Specific TCR and Inhibition of Mispaired-TCR Formation In TCR-Gene Modified T-Cells Can Concomitantly Be Achieved Using a Novel Retroviral Vector with Silencers for Endogenous TCRs

AbstractAbstract 1020 [Purpose]Adoptive engineered T-cell therapy using WT1-specific T-cell receptor gene-transfer is currently considered as a challenging strategy for cancer treatment. However, the formation of shuffled TCR between endogenous and introduced TCR α/β chains still remains a major issue to be solved, not only for the improvement of introduced TCR expression, but also for the prophylaxis of “GVHD-like syndrome” due to newly generated self-reactive T-cells bearing shuffled TCRs. Taking above, we have newly developed an novel WT1-specific TCR retroviral vector with built-in silencers to inhibit endogenous TCRs. In this study, we evaluated the feasibility of this novel WT1-targeting and silencers built-in vector for the clinical application. [Methods]WT1235-243-specific and HLA-A*2402-restricted TCR α/β genes were cloned into this novel retroviral vector with built-in shRNA for endogenous TCRs. (siWT1-TCR) (1) The inhibitory effect on endogenous TCR in gene-modified T-cells was examined using the pre-established CTL clone. (2) Compared with the conventional WT1-TCR vector (without silencers), the functional avidity of siWT1-TCR-gene introduced T-cell was examined, using 51Cr-release assay, CD107a assay and intracellular IFN-γ assay, in vitro. (3) The in vivo anti-leukemia effect of siWT1-TCR introduced CD8+ T-cells was examined using NSG mice. (4) WT1-specific Th1 helper function of siWT1-TCR introduced CD4+ T-cell was examined. (5) The synergistic effect of WT1 peptide stimulation on siWT1-TCR introduced CD8+ T-cell was examined. (6) The on-target adverse effect of siWT1-TCR introduced CD8+ T-cells against autologous hematopoietic progenitor cells were examined using HLA-A*2402+ human cord blood CD34+ and TCR-gene modified autologous CD8+ T-cells, both in vitro and in vivo. [Results]Compared with conventional WT1-TCR vector, siWT1-TCR vector remarkably increased the expression of functional WT1-specific TCR accompanied with the inhibition of endogenous TCR synthesis on gene-modified T-cells. CD8+ and CD4+ T cells engineered with siWT1-TCR gene transfer exerted WT1-specific and leukemia-specific cytotoxicity, and target-responsive Th1 cytokine production, in an HLA-A*2402-restricted fashion, respectively. Mainly because of remarkably increased WT1-specific TCR expression, the anti-leukemia effect exerted by siWT1-TCR introduced CD8+ T-cells was significantly up-regulated, compared with conventional WT1-TCR vector, which reflected the increased the target-responsive granular exocytosis. SiWT1-TCR introduced CD8+ T-cells also exerted in vivo anti-leukemia effect against inoculated leukemia cell lines in NSG mice, furthermore, such siWT1-TCR introduced CD8+ T-cells could kill autologous patients' leukemia cells, but not autologous hematopoietic progenitor cells, in vitro. Eventually, transplanted human hematopoietic progenitor cells which had been precultured with siWT1-TCR introduced autologous CD8+ T-cells into irradiated NSG mice demonstrated that those pre-treated CD34+ cells preserved abilities of engraftment, proliferation and differentiation. Additionally, it was demonstrated that repetitive WT1 peptide stimulations successfully expanded siWT1-TCR introduced CD8+ T-cells, in vitro, which suggests the synergistic effect of combined peptide vaccination in vivo. [Conclusion]Our novel WT1-targeting vector could provide not only the improved functional avidity of gene-modified T-cells, but also a promising option to solve the raised major concern in safety that is the potentially lethal GVHD-like syndrome due to newly generated T-cells bearing self-antigen reactive shuffled TCRs. Based on these pre-clinical observation, we are planning to conduct clinical trials against human hematological malignancies. Disclosures:No relevant conflicts of interest to declare.

Improved Expression and Reactivity of Transduced Tumor-Specific TCRs in Human Lymphocytes by Specific Silencing of Endogenous TCR

Adoptive T-cell therapy using lymphocytes genetically engineered to express tumor antigen-specific TCRs is an attractive strategy for treating patients with malignancies. However, there are potential drawbacks to this strategy: mispairing of the introduced TCR alpha/beta chains with the endogenous TCR subunits and competition of CD3 molecules between the introduced and endogenous TCRs can impair cell surface expression of the transduced TCR, resulting in insufficient function and potential generation of autoreactive T cells. In addition, the risk of tumor development following the infusion of cells with aberrant vector insertion sites increases with the vector copy number in the transduced cells. In this study, we developed retroviral vectors encoding both small interfering RNA constructs that specifically down-regulate endogenous TCR and a codon-optimized, small interfering RNA-resistant TCR specific for the human tumor antigens MAGE-A4 or WT1. At low copy numbers of the integrated vector, the transduced human lymphocytes exhibited high surface expression of the introduced tumor-specific TCR and reduced expression of endogenous TCRs. In consequence, the vector-transduced lymphocytes showed enhanced cytotoxic activity against antigen-expressing tumor cells. Therefore, our novel TCR gene therapy may open a new gate for effective immunotherapy in cancer patients.

Using the hematopoietic transplantation platform for TCR-engineered adoptive immunotherapy of leukemia

Background: Transfer of T cell receptor genes (TCR) directed against tumor-associated antigens can be used to circumvent tolerance and to increase the precursor frequency of tumor-reactive T cells. So far mainly autologous T cells have been used as a T cell source. In a recent clinical trial on melanoma patients (Morgan et. al. Science, 2006) prolonged in vivo survival of the transduced T cells seemed to be critical for a clinical response to therapy. We hypothesized that the use of allogeneic T cells might improve the persistence of TCR gene-transduced T cells after adoptive transfer by a continous alloreactive stimulus mediated through the endogenous TCRs.

Physiological TCR Modulation after Antigen Specific Triggering of Introduced TCRs under Control of a Retroviral Promotor.

TCR transfer to engineer tumor specific T cells may be an alternative strategy for adoptive immunotherapy. We have previously shown that TCR-transduced T cells are capable of recognizing targets both via their endogenous TCR and the introduced TCR. Since the introduced TCR is regulated by a different promotor, we investigated whether triggering and modulation of the introduced TCR occur in a physiological manner compared to the endogenous TCR. Because introduction of a TCRα-chain and TCRβ-chain will lead to formation of chimeric TCR-complexes with the endogenous TCRα- and β-chain, we used models in which both specificities of the TCRs were known and tetramers were available to distinguish each TCR. CMV PP65 specific T cells were transduced with a retroviral construct encoding the hematopoietic minor histocompatibility antigen HA-2 specific TCR (HA-2-TCR). TCR-transduced T cells were antigen specifically triggered using EBV-LCLs that presented endogenously processed PP65- or HA-2-antigen. At various time points after stimulation cell surface expression of the TCRαβ-complexes and the BV-chain of the endogenous TCR was studied with monoclonal antibodies (mAbs). No mAb directed against the BV-chain of the introduced TCR was available. Tetramers specific for the endogenous or introduced TCR were used to distinguish the TCRs from chimeric TCRαβ-complexes. We observed that after stimulation of either the endogenous or the introduced TCR the total amount of TCRs decreased to 40% and 70% of unstimulated cells, respectively. When the endogenous TCR was triggered, HA2- and PP65-tetramer stainings as well as the endogenous BV chain were diminished after 1 day. Stimulation of the introduced TCR decreased the introduced TCR expression after 1 day, and the endogenous TCR expression measured by tetramers and BV specific mAb was marginally decreased. Two days after stimulation of the introduced TCR and 3 days after stimulation of the endogenous TCR, the total amount of TCRαβ-complexes was restored. Staining with specific BV-mAb and tetramers demonstrated that the endogenous TCR expression, both after triggering of the endogenous as well as the introduced TCR, was still decreased at day 3. These data indicate that TCRαβ-complexes on the surface at days 2 and 3 mainly consisted of the introduced HA-2-TCR. TCR expression of non-transduced T cells was still decreased at day 3 after specific stimulation. Functional analysis of the T cells in a chromium release assay after 1 day of TCR triggering demonstrated that the T cells exerted reduced cytolytic activity that correlated with the downmodulation of TCR expression of either the introduced and endogenous TCR. The lytic activity of the T cells was restored at day 2 or 3 and correlated with the tetramer stainings. In conclusion, we observed physiological downmodulation of TCRs which were regulated by a retroviral promotor after antigen specific triggering. However, the introduced TCR was more quickly re-expressed at the cell surface, probably due to the increased retroviral promotor activity. The downmodulation upon specific triggering of both introduced and endogenous TCRs implies that cell mechanisms other than promotor activity are also involved in regulation of TCR cell surface expression.

The Cyclin-Dependent Kinase Inhibitor p27KIP1 Is Mandatory for Tolerance Induction In Vivo.

Elucidating the signals that influence the choice between T cell activation and tolerance in vivo will facilitate the design of novel therapies for the induction of transplantation tolerance. The cyclin-dependent kinase (cdk) inhibitor p27 is an important negative regulator of cell cycle progression in T lymphocytes. We have previously observed that in vitro generated anergic T cells express high levels of p27 and that forced expression of p27 renders T cells incapable of progressing through the cell cycle and transcribing the IL-2 gene. Therefore, we sought to determine the role of p27 on the decision of naive T cells between activation and tolerance in vivo. In order to eliminate the ability of p27 to regulate the cell cycle without disrupting other interactions of the protein, we used mice in which only the cdk-binding domain of p27 has been deleted resulting in a truncated form of p27 (p27tr). To generate a T cell population, which uniformly expresses a specific TCR that allows antigen-specific stimulation in vivo, we bred the DO11.10 TCR transgene onto the p27tr background. DO11.10 mice express a MHC class II-restricted transgenenic (Tg) TCR specific for OVA323–339 peptide that can be detected by a clonotypic antibody. To generate animals with T cells exclusively expressing the DO11.10 TCR, we crossed the DO11.10 TCR-Tg mice onto RAG2−/− background which eliminates endogenous TCRs. Naive T cells from DO11.10+RAG2−/−p27tr mice (termed p27tr/Tg) expressed markers identical to those of naive T cells from wild type DO11.10+RAG2−/− (termed WT-Tg) mice, allowing the accurate comparison of naive cells that differ only in their ability to express the p27 cdk-binding domain. Tg T cells from WT-Tg or p27tr/Tg mice were adoptively transferred into syngeneic recipients, which were injected with OVA323–339 either subcutaneously (primed) or intravenously (tolerized) according to established protocol. Draining lymph nodes were harvested and Tg cells were stimulated with OVA323–339 to determine their ability to respond on antigen-specific rechallenge. WT-Tg and p27tr/Tg cells from the primed treatment group had robust proliferation and production of IL-2. Strikingly, in the tolerant treatment group, although WT-Tg cells proliferated poorly and had significantly reduced IL-2 production, p27tr/Tg cells had proliferation and IL-2 production that were indistinguishable from the p27tr/Tg primed treatment group. Next we used blockade of costimulation to induce tolerance in vivo. Tg T cells from WT-Tg or p27tr/Tg mice were adoptively transferred into syngeneic recipients, which were treated with either PBS (primed) or with anti-CD40L mAb plus CTLA4-Ig (tolerized) and were immunized with OVA323–339 subcutaneously. Subsequent rechallenge with OVA323–339 revealed that, after in vivo treatment of anti-CD40L mAb and CTLA4-Ig, only WT-Tg cells became tolerant and were incapable of proliferating and producing IL-2, whereas responses of p27tr/Tg cells were not suppressed. Detailed analysis revealed that the failure of p27tr/Tg cells to undergo tolerance induction in the syngeneic recipients was not due to defective suppression by Treg, but due to the intrinsic inability of p27tr/Tg T cells to undergo arrest at the G1 phase of the cell cycle during tolerogenic treatment. These results demonstrate a key role for p27 in determining the outcome of naive T cell encounter with a tolerogenic stimulus in vivo and have significant implications for developing of immune based therapies geared towards tolerance induction.

A Defect in Deletion of Nucleosome-Specific Autoimmune T Cells in Lupus-Prone Thymus: Role of Thymic Dendritic Cells

To study central tolerance to the major product of ongoing apoptosis in the thymus, we made new lines of transgenic (Tg) mice expressing TCR of a pathogenic autoantibody-inducing Th cell that was specific for nucleosomes and its histone peptide H4(71-94). In the lupus-prone (SWR x NZB)F1 (SNF1) thymus, introduction of the lupus TCR transgene caused no deletion, but marked down-regulation of the Tg TCR and up-regulation of endogenous TCRs. Paradoxically, autoimmune disease was suppressed in the alphabetaTCR Tg SNF1 mice with induction of highly potent regulatory T cells in the periphery. By contrast, in the MHC-matched, normal (SWR x B10. D2)F1 (SBF1), or in the normal SWR backgrounds, marked deletion of transgenic thymocytes occurred. Thymic lymphoid cells of the normal or lupus-prone mice were equally susceptible to deletion by anti-CD3 Ab or irradiation. However, in the steady state, spontaneous presentation of naturally processed peptides related to the nucleosomal autoepitope was markedly greater by thymic dendritic cells (DC) from normal mice than that from lupus mice. Unmanipulated thymic DC of SNF1 mice expressed lesser amounts of MHC class II and costimulatory molecules than their normal counterparts. These results indicate that apoptotic nucleosomal autoepitopes are naturally processed and presented to developing thymocytes, and a relative deficiency in the natural display of nucleosomal autoepitopes by thymic DC occurs in lupus-prone SNF1 mice.

The Functional Activity of Genetically Engineered T Cell Receptor Transferred T Cells Is Highly Dependent on Pairing Properties of the Transferred TCR α and β Chains.

The genetic engineering of T lymphocytes is an attractive strategy to specifically redirect T cell immunity towards viral infections and malignancies. Transfer of virus- or tumor-specific TCRs has demonstrated to endow T cells with redirected antigen specificity. We demonstrated redirected anti-leukemic reactivity of CMV specific T cells using gene transfer of minor histocompatibility antigen HA-2 specific TCRs. The HA-2-TCR-modified T cells exerted high cytolytic activity against HA-2 expressing target cells, including leukemic cells, and not against target cells negative for the HA-2 mHag. After cloning of the TCR-transferred T cells, we demonstrated that the HA-2-TCR cell surface expression, measured by HA-2-tetramer staining, was variable on the transduced T cell clones, and that the cytolytic capacity of the T cells correlated with the level of HA-2-TCR expression.Since we could demonstrate that this variation in HA-2-TCR expression was not due to differences in transgene expression, we investigated whether the endogenous TCRs influenced the expression of the introduced TCR. CMV-A2 specific T cells were isolated from peripheral blood and transduced with the HA-2-TCR. In control transduced CMV specific T cells we observed 5 different high affinity CMV specific TCRs. CMV specific T cells transduced with the HA-2-TCR that expressed predominantly the HA-2-TCR, expressed only one of these types of CMV-TCR, and in CMV specific T cells with low HA-2-TCR expression two different types of CMV-TCRs were found. These data indicated that the level of expression of the introduced TCR is strongly influenced by the endogenous TCR. To investigate whether this was due to differences in promotor activity of the endogenous and retrovirally introduced TCR, the three CMV-TCRs were characterized and transferred into unselected peripheral T cells. T cells transferred with the weak competitior CMV-TCR that was strongly downregulated in CMV specific T cells by introduction of the HA-2-TCR, showed low CMV specific cytotoxicity and no tetramer staining. In contrast, T cells transferred with the strong competitor CMV-TCR that was modestly downregulated in CMV specific T cells by introduction of the HA-2-TCR, revealed strong CMV specific cytotoxic activity and tetramer staining. These data demonstrate that the introduced and endogenous TCRs compete for cell surface expression, and that this competition is dependent on characteristics of the different TCRs and independent of whether the TCR is retrovirally introduced or naturally expressed. To investigate whether the cell surface expression of the different TCRs was determined by preferential pairing properties of the individual TCR chains, TCR α and β deficient Jurkat 76 cells were transduced with the three CMV-specific TCRs or with chimeric TCRs consisting of the TCR α chain of one TCR with the TCR β chain of another TCR. TCRαβ membrane expression revealed that TCRs with a strong competitor phenotype expressed higher levels of TCRαβ than the TCR that was a weak competitor. TCRαβ expression of Jurkat cells transduced with chimeric TCRs indicated that the expression level of the different TCRs was determined by the pairing properties of the individual TCR α and β chains and not by differences in protein expression. In conclusion these data demonstrated that introduced and endogenous TCRs compete for cell surface expression in favor of the TCR that has the highest intrinsic pairing properties.