TCR Gene Therapy Research Articles

More T cell receptors to the RAScue in cancer?

Treatment with T cells genetically engineered to express tumor-reactive T cell receptors (TCRs), known as TCR-gene therapy (TCR-T), is a promising immunotherapeutic approach for patients with cancer. The identification of optimal TCRs to use and tumor antigens to target are key considerations for TCR-T. In this issue of the JCI, Bear and colleagues report on their use of in vitro assays to characterize four HLA-A*03:01- or HLA-A*11:01-restricted TCRs targeting the oncogenic KRAS G12V mutation. The TCRs were derived from healthy donors or patients with pancreatic cancer who had received a vaccine against mutant KRAS. The most promising TCRs warrant testing in patients with KRAS G12V-positive cancers.

Expanding the landscape of TCR gene therapy targeting MAGE

Expanding the landscape of TCR gene therapy targeting MAGE

Process intensification for lentiviral vector manufacturing using tangential flow depth filtration

For gene therapies to become more accessible and affordable treatment options, process intensification is one possible strategy to increase the number of doses generated per batch of viral vector. Process intensification for lentiviral vector manufacturing can be achieved by enabling perfusion in the production bioreactor when applied in tandem with a stable producer cell line, allowing for significant expansion of cells and production of lentiviral vectors without the need for transfer plasmids. Tangential flow depth filtration was used to achieve an intensified lentiviral vector production by enabling perfusion to expand cell density and allow for continuous separation of lentiviral vectors from producer cells. Hollow-fiber depth filters made of polypropylene with 2- to 4-μm channels demonstrated high filter capacity, extended functional life, and efficient separation of lentiviral vectors from producer cells and debris when used for this intensified process. We anticipate that process intensification with tangential flow depth filtration at 200-L scale from a suspension culture can produce on the order of magnitude of 10,000 doses per batch of lentiviral vectors required for CAR T or TCR cell and gene therapy that would require approximately 2×109 transducing units per dose.

PIK3CA hotspot mutation generates a shared neoantigen targetable by TCR gene therapy

PIK3CA hotspot mutation generates a shared neoantigen targetable by TCR gene therapy

Non-Mutated Nucleophosmin 1 Is Recognized by the CD8+ T Lymphocytes of an AML Patient after the Transplantation of Hematopoietic Stem Cells from an HLA-Haploidentical Donor.

Simple SummaryOur study describes an AML patient whose leukemia cells carried the NPM1c+ mutation, and who was the recipient of allogeneic HSCT from a haploidentical donor. The patient raised a robust allorestricted CD8+ T cell response directed against the NPM1wt protein. Favourably, the response against NPM1wt was not accompanied by side effects such as GvHD. Moreover, the induction of a high NPM1wt specific response coincided with the decrease in NPM1c+ transcripts detected, implying a beneficial graft versus leukemia effect. On the basis of these results, we suppose that TCRs from allorestricted NPM1wt-specific T cells are worth studying in other recipients of grafts from haploidentical donors as a possible tool for TCR gene therapy.Nucleophosmin (NPM1, B23) is a multifunctional phosphoprotein expressed in all tissues. The protein is mainly localized in nucleoli. In hematological malignancies, NPM1 belongs to commonly altered genes. Its mutation, always heterozygous, leads to the re-localization of the NPM1 protein from the nucleolus to the cytoplasm (NPM1c+). NPM1c+ is found in 30% of acute myeloid leukemia (AML). Our study showed that an AML patient, whose leukemia cells carried the NPM1c+ mutation and who was the recipient of allogeneic HSCT from a haploidentical donor, raised a robust allorestricted CD8+ T cell response directed against the NPM1wt protein. Favourably, the response against NPM1wt was not accompanied by side effects such as GvHD. Moreover, the induction of a high NPM1wt-specific response coincided with the decrease in NPM1c+ transcripts detected, implying a beneficial graft versus leukemia effect. On the basis of these results, we suppose that TCRs from allorestricted NPM1wt-specific T cells are worth studying in other recipients of grafts from haploidentical donors as a possible tool for TCR gene therapy.

TCR Gene Therapy for Cancer.

The protocol describes the procedure of antigen-specific T cell generation and TCR identification for the use in adoptive T cell therapy. We describe two paths of generating antigen-specific T cells, first, T cell stimulation with autologous dendritic cells pulsed with antigen peptide, second, in vivo T cell stimulation with peptide or DNA by gene gun application in a suitable mouse model followed by in vitro enrichment of peptide-reactive T cells. Peptide-stimulated T cells are sorted by fluorescence-activated cell sorting for CD107α or IFNγ expression and subsequently isolated RNA is used in a 5' rapid amplification of cDNA ends (RACE )-PCR specific for TCR for TCR chain identification. After retroviral cloning, it is re-expressed on human T cells to test its applicability in adoptive T cell therapy.

Development of T-Cell Therapy By Exploiting Modified Antibodies Specific for A2/NY-ESO-1 for Refractory Myeloma

Background: Clinical trials have demonstrated that T-cell-based immunotherapy can suppress tumor growth even in patients with refractory malignancies. Multiple myeloma (MM) still remains incurable and many patients experience disease progression even though novel therapeutic agents have been developed. NY-ESO-1 is a well-known cancer-testis antigen which is expressed by myeloma cells in about half of MM patients, especially in aggressive cases, and NY-ESO-1157-165 peptide presented by an HLA-A*02:01 molecule (A2/NY-ESO-1157) on myeloma cell surfaces have been shown. In a recent clinical trial, adoptive therapy using T cells modified with T-cell receptor (TCR) specific for A2/NY-ESO-1157 in combination with autologous stem cell transplantation successfully induced clinical responses in 80% patients with advanced MM. However, generation of TCR-transduced T cells are laborious, and potentially bearing a risk of lethal adverse events due to the cross-reactivity of expressing TCR. Modified antibodies; chimeric antigen receptor (CAR) and bispecific antibody (BsAb) which mediate antitumor T-cell responses have shown promise in clinical trials. We hypothesized that these two modalities enable to expand the clinical versatility of T-cell therapy targeting NY-ESO-1 in the treatment of refractory myeloma. In this study, we have generated both CAR and BsAb which recognize A2/NY-ESO-1157, and assessed their anti-myeloma reactivity in vitro .Methods: Based on the structure of previously reported monoclonal antibody specific for A2/NY-ESO-1157 (clone: 3M4E5), we newly synthesized two different A2/NY-ESO-1157-specific single chain fragment variable (scFv) genes encoding variable regions of a light (L) and a heavy (H) chain in order, and vice versa (HL). Second generation CAR with each scFv linked with CD28 and CD3ζ was generated. A BsAb composed of each A2/NY-ESO-1157-specific scFv and a CD3ε-reactive scFv which can stimulate peripheral T cells was also generated. A2/NY-ESO-1157-specific reactivity mediated by CAR-T cells were assessed by A2/NY-ESO-1157 tetramer and multiple cytokine assays. Target-specific cytokine production induced by peripheral T cells in the presence of BsAbs was similarly examined. Specific lysis of target cells mediated by T cells redirected with modified antibodies was measured by standard 51Cr-release assay. Expression of NY-ESO-1 in a panel of myeloma cell lines was examined by real-time PCR and western blotting.Results: Three out of six myeloma cell lines we tested abundantly expressed NY-ESO-1 mRNA and protein. CAR-transduced T cells established from five out of five donors showed A2/NY-ESO-1157-specific reactivity. These gene-modified T cells recognized and killed target cells which naturally process and present A2/NY-ESO-1157 on their cell surfaces, resulting in anti-myeloma reactivity to A2+NY-ESO-1+ U266 myeloma cells. LAGE-1 is a well-known homolog of NY-ESO-1, and fully shares the sequence of NY-ESO-1157-165 peptide. As expected, CAR-T cells recognized and killed A2+LAGE-1+ target cells. Newly generated BsAbs successfully linked A2/NY-ESO-1157 expressing targets and CD3+ T cells. Functional avidity of LH-BsAb-stimulated peripheral T cells for A2/NY-ESO-1157 was higher than that of T cells stimulated with HL-BsAb, and was comparable with that mediated by CAR-T cells. BsAb-stimulated T cells produced multiple cytokines against both A2+NY-ESO-1+ and A2+LAGE-1+ target cells, and lysed both of them. Importantly, compared with CAR-T cells, BsAb-stimulated T cells showed the superior A2/NY-ESO-1157-reactive cytokine production capacity.Conclusions: T cells redirected with CAR and BsAb both successfully showed anti-myeloma reactivity in an A2/NY-ESO-1157-specific manner. An A2/NY-ESO-1157-reactive BsAb displayed a potential to induce sufficient antitumor T-cell responses against myeloma cells. In addition to TCR-gene therapy targeting NY-ESO-1, these two scFv-based modalities would be able to provide efficacious and flexible options for the treatment of patients with refractory MM. DisclosuresNo relevant conflicts of interest to declare.

The First Functional HLA-a*01:01-Restricted EBV-LMP2-Specific T-Cell Receptors for TCR Gene Therapy of Patients with EBV-Associated Type II/III Malignancies

Epstein Barr virus (EBV) is associated with the development of a broad range of malignancies, including Burkitt's lymphoma, Hodgkin and non-Hodgkin lymphomas, post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma and gastric carcinoma. Differential expression of immunogenic antigens (e.g. EBV Nuclear Antigen (EBNA2-6) and Latent membrane proteins (LMPs)) is seen at the different latent phases of the viral infection. Although many EBV-associated lymphomas only express weakly immunogenic EBV antigens (e.g. EBNA1 and BARF1), lymphomas with type II or III latency express LMP1 and LMP2. Growth of such lymphomas can be curbed using adoptively transferred EBV-LMP1/2-specific T cells. Surprisingly, T cells recognizing EBV-derived peptides in the common HLA allele A*01:01 have not been found. In addition, an HLA-A*01:01-associated increased risk for EBV+ Hodgkin lymphomas and infectious mononucleosis has been reported, suggesting that HLA-A*01:01-restricted EBV-specific T cells may be absent or present at very low frequencies in these patients. A need thus exists for HLA-A*01:01-restricted EBV-specific T-cell products, especially directed against EBV-LMP1/2. Based on MHC class I peptide predictions, HLA-A*01:01-binding peptides derived from different immunogenic EBV antigens were identified and tetramer complexes were synthesized (EBNA3A-YTDHQTTPT, EBNA3A-FLQRTDLSY, BZLF1-FTPDPYQVPF, LMP2-ESEERPPTPY, LMP2-LTEWGSGNRTY). HLA-A*01:01-restricted EBV-specific T cells were present at very low frequencies in total PBMCs from all 6 donors. After sorting using flow cytometry, only EBV-LMP2-ESE specific T cells could be expanded (for 5/6 donors), yielding pure tetramer+ CD8 T-cell populations. Four out of 5 isolated T-cell populations exhibited intermediate to high avidity recognition of HLA-A*01:01-transduced TAP2-deficient T2 cells, loaded with EBV-LMP2-ESE peptide. This specific LMP2-derived peptide showed to be functionally processed, presented and recognized by EBV-LMP2-ESE-specific T cells when using HLA-A*01:01/LMP2-transduced K562 cells. To assess the suitability for TCR gene-therapy, the TCRs from the 4 functional T-cell populations were sequenced and cloned into a retroviral vector. Surprisingly, all 4 EBV-LMP2-ESE-specific T-cell populations used the TRBV6-2 gene for TCR beta-chain expression. Additionally, for TCRalpha-chain expression these populations used either TRAV12 or TRAV30. These TCRs contained small differences in the CDR3 region. Despite differences in tetramer binding, all TCRs were functional when transduced into primary CD8 T cells, CD4 T cells, and CD8 negative TCR knock-out Jurkat cells, implying CD8 independent recognition. Finally, recognition of HLA-A*01:01+ EBV-LCLs demonstrated the potential of these EBV-LMP2-ESE-specific TCRs to recognize naturally occurring endogenous LMP2. In conclusion, we isolated and validated the first functional HLA-A*01:01-restricted EBV-LMP2-specific T-cell populations and TCRs, which can be used for adoptive transfer or retro/lentiviral TCR gene therapy to treat EBV-associated type II/III lymphomas, EBV+ malignancies of epithelial origin and PTLDs. Disclosures No relevant conflicts of interest to declare.

Simultaneous Deletion of Endogenous TCRαβ for TCR Gene Therapy Creates an Improved and Safe Cellular Therapeutic.

Generation of an optimal Tcell therapeutic expressing high frequencies of transgenic Tcell receptor (tgTCR) is essential for improving TCR gene therapy. Upon TCR gene transfer, presence of endogenous TCRαβ reduces expression of tgTCR due to TCR mixed-dimer formation and competition for binding CD3. Knockout (KO) of endogenous TCRαβ was recently achieved using CRISPR/Cas9 editing of the TRAC or TRBC loci, resulting in increased expression and function of tgTCR. Here, we adopt this approach into current protocols for generating Tcell populations expressing tgTCR to validate this strategy in the context of four clinically relevant TCRs. First, simultaneous editing of TRAC and TRBC loci was reproducible and resulted in high double KO efficiencies in bulk CD8 Tcells. Next, tgTCR expression was significantly higher in double TRAC/BC KO conditions for all TCRs tested, including those that contained structural modifications to encourage preferential pairing. Finally, increased expression of tgTCR in edited Tcell populations allowed for increased recognition of antigen expressing tumor targets and prolonged control of tumor outgrowth in a preclinical model of multiple myeloma. In conclusion, CRISPR/Cas9-mediated KO of both endogenous TCRαβ chains can be incorporated in current Tcell production protocols and is preferential to ensure an improved and safe clinical therapeutic.

Framework engineering to produce dominant T cell receptors with enhanced antigen-specific function

TCR-gene-transfer is an efficient strategy to produce therapeutic T cells of defined antigen specificity. However, there are substantial variations in the cell surface expression levels of human TCRs, which can impair the function of engineered T cells. Here we demonstrate that substitutions of 3 amino acid residues in the framework of the TCR variable domains consistently increase the expression of human TCRs on the surface of engineered T cells.The modified TCRs mediate enhanced T cell proliferation, cytokine production and cytotoxicity, while reducing the peptide concentration required for triggering effector function up to 3000-fold. Adoptive transfer experiments in mice show that modified TCRs control tumor growth more efficiently than wild-type TCRs. Our data indicate that simple variable domain modifications at a distance from the antigen-binding loops lead to increased TCR expression and improved effector function. This finding provides a generic platform to optimize the efficacy of TCR gene therapy in humans.

Abstract 1429: Preclinical characterization of a KK-LC-1-specific T cell receptor for the treatment of epithelial cancers

Abstract T cell receptor (TCR) gene therapy can mediate tumor regression in patients; however, successful treatment of epithelial cancers has been limited. We studied the targeting of Kita-Kyushu lung cancer antigen 1 (KK-LC-1), a cancer germline antigen expressed in multiple epithelial cancers, with TCR gene-engineered T cells. We thoroughly vetted KK-LC-1 expression in both cancerous and healthy tissue by pulling data from BioGPS and cBioPortal and analyzing a broad range of patient samples, primary cancer lines, and healthy tissue by both qRT-PCR and fluorescence in situ hybridization. Our findings show that KK-LC-1 is expressed in numerous epithelial cancers including breast, lung, cervical, ovarian, melanoma, and prostate whereas expression in healthy tissue is limited to immune-privileged sites including the epididymis and testis. We identified an HLA-A*01:01-restricted KK-LC-152-60-specific TCR isolated from the tumor infiltrating lymphocytes of a patient with metastatic cervical adenocarcinoma. T cells from two healthy donors were transduced with the KK-LC-1 TCR and tested for in vitro targeting of tumor cell lines. The KK-LC-1 TCR recognized the HLA-A*01:01+, KK-LC-1+ cell lines 4156, EKVX, and PC-3, as determined by IFN-γ release. The HLA-A*01:01- cell lines, HeLA, DU-145, and MDA-MD-468, were initially not recognized by the KK-LC-1 TCR; however, following stable transduction with HLA-A*01:01, these cell lines were targeted. The HLA-A*01:01+, KK-LC-1- cell lines, PC-3 and 3748, were not recognized by the KK-LC-1 TCR. To assess the TCR’s potential for off-target activity, we used amino acid scanning to determine the recognition motif, allowing for subsequent targeted in silico searches to identify peptides containing the same recognition motif. Using NCBI Blast and the ScanProsite database, 10 peptides with homologous essential but different non-essential residues were identified. A retroviral vector encoding the KK-LC-152-60 TCR was used to genetically engineer peripheral blood T cells. No peptide recognition by the TCR was observed when dendritic cells were pulsed with the individual peptides and co-cultured with KK-LC-1 TCR-transduced T cells. Finally, we assessed if human genetically engineered T cells expressing the KK-LC-1 TCR could mediate in vivo tumor regression in an NSG mouse model. Genetically engineered T cells expressing the KK-LC-1 TCR induced tumor regression in both a melanoma and cervical cancer line in a dose-dependent manner. Collectively, these findings indicate that KK-LC-1 is a suitable target for TCR gene therapy and they provide preclinical support for testing of the KK-LC-1 TCR in a phase I clinical trial. Citation Format: Bridget Marcinkowski, Carylinda Serna, Benjamin Jin, Scott Norberg, Christian Hinrichs. Preclinical characterization of a KK-LC-1-specific T cell receptor for the treatment of epithelial cancers [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 1429.

Abstract B205: Adoptive transfer of autologous T-cells, modified with a MART-1 specific TCR and cultured in IL-7/IL-15, for the treatment of metastatic melanoma patients

Abstract Currently a TCR gene therapy trial to treat stage IV cutaneous and uveal melanoma patients is being conducted at the NKI. The TCR used for T-cell modification is specific for the HLA-A*0201 restricted MART-126-35 epitope, which is expressed on the majority of melanoma cells. Unique to this trial is the use of the combination of anti-CD3/CD28 beads for T-cell activation plus IL-7/ IL-15 for subsequent culture and expansion, instead of the more commonly used strategy that utilizes the combination of anti-CD3 and IL-2. The aim of this alternative production strategy is to generate a “less differentiated“ T-cell product that may have a better engraftment potential and antitumor activity. Thus far, a total of 12 patients have been treated, of whom 5 were uveal melanoma patients. 1 patient treated with 5x109 cells died 9 days after infusion, among others due to a severe cytokine release syndrome. The other 11 patients were treated in 3 different cell doses: 5x107, 25x107and10x107 transduced CD4 and CD8 T-cells. A prolonged engraftment and higher % of gene modified cells in the blood compartment were observed in patients treated with the highest number of cells infused. Gene modified T-cells could be found back in the circulation for up to two months post infusion, even when as few as 5x107 transduced T-cells were infused. In the highest cell dose, transduced T-cells could be detected up to 10 months following infusion. On-target toxicity against MART-1 expressing melanocytes in the skin (rash and vitiligo) was observed in all patients. Most severe skin toxicity, but also uveitis and inner ear toxicity, were observed in patients treated in the highest dose cohort of 2.5x107 transduced T-cells. These patients also developed a cytokine release syndrome requiring tocilizumab (anti-IL6R) treatment. Unexpected toxicity observed at this highest dose level consisted of development of pancytopenia around 6-8 weeks post T-cell infusion. In one of these patients, T-cell clonality of the infused cell product and of the peripheral blood compartment at the time of pancytopenia was analyzed in order to investigate the mechanism behind this toxicity. The highest clonality was found in the endogenous unmodified CD4 population. TCR diversity and evenness of the CD8 modified and unmodified populations were similar, suggesting that the pancytopenia was not associated with specific expansion of a TCR modified T-cell clone. However, this delayed pancytopenia could be related to 1) the conditioning chemotherapy regimen (fludarabine and cyclophosphamide), 2) cytokine release like IL-6, or 3) expansion of an autoreactive clone derived either from the infused T-cell product or newly generated from the endogenous T-cell pool. Of the 12 patients treated thus far, 8 patients had stable disease (SD) and two showed a partial response (PR) (5.2 and 9.5 month). The type of response or the duration of response was not correlated to the amount of cells infused or the peak and duration of engraftment of the infused T-cells. On the basis of these preliminary data, we conclude that TCR-modified T-cells produced by this method have a high engraftment potential and show in vivo activity at low infused cell doses, thereby suggesting the potential value of this protocol for TCR or CAR gene therapy for the treatment of cancer. Citation Format: Raquel Gomez-Eerland, Maartje Rohaan, Joost van den Berg, Maaike van Zon, Renate de Boer, Noor Bakker, Bastiaan Nuijen, Ton N.M. Schumacher, John B.A.G. Haanen. Adoptive transfer of autologous T-cells, modified with a MART-1 specific TCR and cultured in IL-7/IL-15, for the treatment of metastatic melanoma patients [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 B205.

PRAME and HLA Class I expression patterns make synovial sarcoma a suitable target for PRAME specific T-cell receptor gene therapy

ABSTRACTSynovial sarcoma expresses multiple cancer testis antigens that could potentially be targeted by T-cell receptor (TCR) gene therapy. In this study we investigated whether PRAME-TCR-gene therapy could be an effective treatment for synovial sarcoma by investigating the potential of PRAME-specific T-cells to recognize sarcoma cells and by evaluating the expression patterns of PRAME and HLA class I (HLA-I) in synovial sarcoma tumor samples. All PRAME expressing sarcoma cell lines, including 2 primary synovial sarcoma cell cultures (passage < 3), were efficiently recognized by PRAME-specific T-cells. mRNA FISH demonstrated that PRAME was expressed in all synovial sarcoma samples, mostly in an homogeneous pattern. Immunohistochemistry demonstrated low HLA-I baseline expression in synovial sarcoma, but its expression was elevated in specific areas of the tumors, especially in biphasic components of biphasic synovial sarcoma. In 5/11 biphasic synovial sarcoma patients and in 1/17 monophasic synovial sarcoma patients, elevated HLA-I on tumor cells was correlated with infiltration of T-cells in these specific areas. In conclusion, low-baseline expression of HLA-I in synovial sarcoma is elevated in biphasic areas and in areas with densely infiltrating T-cells, which, in combination with homogeneous and high PRAME expression, makes synovial sarcoma potentially a suitable candidate for PRAME-specific TCR-gene therapy.

Isolation of T cell receptors targeting recurrent neoantigens in hematological malignancies

Mutation-derived neoantigens represent an important class of tumour-specific, tumour rejection antigens, and are attractive targets for TCR gene therapy of cancer. The majority of such mutations are patient-specific and targeting these requires a fully personalized approach. However, some mutations are found recurrently among cancer patients, and represent potential targets for neoantigen-specific TCR gene therapy that is more widely applicable. Therefore, we have investigated if some cancer mutations found recurrently in hematological malignancies encode immunogenic neoantigens presented by common European Caucasoid HLA class I alleles and can form targets for TCR gene therapy. We initially focused on identifying HLA class I neoepitopes derived from calreticulin (CALR) exon 9 mutations, found in ~ 80% of JAK2wt myeloproliferative neoplasms (MPN). Based on MHC class I peptide predictions, a number of peptides derived from mutant CALR (mCALR) were predicted to bind to HLA-A*03:01 and HLA-B*07:02. However, using mass spectrometry and ex vivo pMHC multimer staining of PBMC from MPN patients with CALR exon 9 mutations, we found no evidence that these peptides were naturally processed and presented on the surface of mCALR-expressing target cells. We next developed a protocol utilizing pMHC multimers to isolate CD8+ T cells from healthy human donor PBMC that are specific for mCALR and additional putative neoepitopes found recurrently in hematological malignancies. Using this approach, CD8+ T cells specific for HLA-A*03:01- and HLA-B*07:02-presented mCALR peptides and an HLA-A*11:01-presented mutant FBXW7 (mFBXW7) peptide were successfully isolated. TCRs isolated from mCALR-specific CD8+ T cell populations were not able to recognize target cells engineered to express mCALR. In contrast, mFBXW7-specific CD8+ T cells were able to recognize target cells engineered to express mFBXW7. In conclusion, while we found no evidence for mCALR derived neoepitope presentation in the context of the HLA class I alleles studied, our data suggests that the recurrent pR465H mutation in FBXW7 may encode an HLA-A*11:01 presented neoepitope, and warrants further investigation as a target for T cell based immunotherapy of cancer.

Detection of clinically relevant T-cell receptors requires tailored approaches, and TCR gene therapy carries inherent risks

Detection of clinically relevant T-cell receptors requires tailored approaches, and TCR gene therapy carries inherent risks

Characterisation of gene-modified CD3-enhanced CD4+ T cells for cancer immunotherapy

Abstract TCR gene transfer is used to redirect the antigen specificity of T lymphocytes towards known tumour antigens. TCR gene therapies in murine studies have shown promising results. However, in the clinic they often generate sub-optimal responses, when compared to treatments with tumour infiltrating lymphocytes. Previous work to improve TCR gene therapy has demonstrated that transferring additional CD3 genes increases TCR expression of both endogenous and introduced TCR in CD4+ and CD8+ T cells. In vivo experiments demonstrated that CD8+ T cells, transduced with TCR and additional CD3 were more effective in tumour protection than T cells transduced with the TCR alone. Whilst, CD4+ T cells transduced with TCR and additional CD3 initially showed improved tumour protection, lethal toxicity, unrelated to tumour burden, was later observed. To investigate the effects of CD3 overexpression, CD3 genes only (no TCR genes) were transferred into purified CD4+ and CD8+ T cells. Following adoptive transfer, CD3-enhanced CD4+ T cells survived for longer and were recovered in higher percentages in spleen, lymph nodes, bone marrow and liver, compared to CD3-enhanced CD8+ T cells and mock transduced CD4+ T cells. The same trend was also seen in competition experiments where mice received a 1:1 ratio of CD3-enhanced CD4+ T cells and mock-transduced CD4+ T cells. Interestingly, this was observed despite a twofold downregulation of TCR levels in the CD3-enhanced CD4+ T cells, compared to their pre-transfer TCR levels. Current experiments are aimed at dissecting the mechanisms responsible for, and the physiological implications of the observed TCR downregulation.

Tumor- and Neoantigen-Reactive T-cell Receptors Can Be Identified Based on Their Frequency in Fresh Tumor.

Adoptive transfer of T cells with engineered T-cell receptor (TCR) genes that target tumor-specific antigens can mediate cancer regression. Accumulating evidence suggests that the clinical success of many immunotherapies is mediated by T cells targeting mutated neoantigens unique to the patient. We hypothesized that the most frequent TCR clonotypes infiltrating the tumor were reactive against tumor antigens. To test this hypothesis, we developed a multistep strategy that involved TCRB deep sequencing of the CD8(+)PD-1(+) T-cell subset, matching of TCRA-TCRB pairs by pairSEQ and single-cell RT-PCR, followed by testing of the TCRs for tumor-antigen specificity. Analysis of 12 fresh metastatic melanomas revealed that in 11 samples, up to 5 tumor-reactive TCRs were present in the 5 most frequently occurring clonotypes, which included reactivity against neoantigens. These data show the feasibility of developing a rapid, personalized TCR-gene therapy approach that targets the unique set of antigens presented by the autologous tumor without the need to identify their immunologic reactivity. Cancer Immunol Res; 4(9); 734-43. ©2016 AACR.

Expanding the scope of WT1- and cyclin A1-specific TCR gene therapy for AML and other cancers

Abstract TCR gene therapy hastens the reproducible generation of tumor-specific T cells from patients, creating a promising avenue for treating hematological and solid tumors. Several hurdles remain to making this a broadly effective modality, including variable target antigen expression, risk of tumor antigenic escape, and current therapies limited almost exclusively to HLA-A0201+ patients. We previously characterized WT1 and cyclin A1 as favorable T cell immunotherapy targets, based on high expression in malignant versus normal cells and roles in oncogenesis, reducing likelihood of loss variants. We currently have a high-affinity HLA-A0201/WT1-specific TCR in Phase I trials for the treatment of AML and NSCLC, and are now focused on developing WT1- and cyclin A1-specific TCRs for ~7 common HLA types, aiming to cover &amp;gt;90% of patients. T cell lines were generated by stimulating donor PBMC with overlapping peptide libraries to identify new reactive epitopes, and the highest affinity T cell clones for each HLA/antigen combination identified by tetramer binding strength. TCRs were then cloned into lentiviral vectors for expression in CD8 T cells. We are currently in the process of selecting the best HLA-A0201/cyclin A1-specific TCR with which to proceed to clinical development. We are also continuing to optimize our epitope discovery protocol for the improved selection of TCRs for other HLA alleles. Safety and efficacy of selected TCRs are rigorously tested using bioinformatic screens, in vitro assays, and humanized MHC-I mouse models. Our goal is to create a molecular toolbox of off-the-shelf TCR reagents to facilitate rapid treatment of patients, using T cell immunotherapies targeted to their particular tumor antigenic profile and HLA type.

272. Rapid Identification of Tumor- and Neoantigen-Reactive T-Cell Receptors for Personalized Immunotherapy

Adoptive transfer of T-cell receptor (TCR) gene engineered T-cells targeting tumor-associated antigens can mediate cancer regression. Accumulating evidence suggests that the clinical success of many immunotherapies is mediated by T-cells targeting mutated neoantigens unique to the patient. We hypothesized that the most frequent TCR clonotypes infiltrating the tumor were reactive against tumor antigens. We thus developed a multi-step strategy that involved CDR3-TCRB deep sequencing of tumor infiltrating lymphocytes and matching of TCRA-TCRB pairs by pairSEQ and single cell RT-PCR. CDR3 TCRA-TCRB pairs identified with this approach, were then reconstructed to full length based on IMGT database [http://www.imgt.org], synthetized and cloned into non-viral and retroviral expression vectors. T-cells were gene-engineered with these TCRs and tested against tumor cell lines and antigen presenting cells expressing tandem minigenes encoding mutated tumor neoantigens and/or pulsed with corresponding mutated peptides. Analysis of 12 fresh metastatic melanomas revealed that in 11 samples, up to 5 tumor-reactive TCRs were present in the 5 most frequently occurring clonotypes including reactivity against neoantigens. These data demonstrate the feasibility of developing a rapid approach for the identification of tumor-reactive TCRs that can be used for personalized TCR-gene therapy.

Abstract A85: Identification and enrichment for rare naturally occurring tumor-reactive T cells in the tumor microenvironment reveals new and diverse opportunities for cancer immunotherapy

Abstract The existence of naturally-occurring, tumor-reactive T-cells in human cancer has rationalized the application of immunotherapy in oncology, however the immunobiology of spontaneous tumor-reactive T-cells in cancer is not well-defined due to difficulties in identifying and validating these responses. The ability to identify and characterize rare tumor-reactive T cells has strong prognostic value and holds the potential to enable various forms of immunotherapy, including adoptive T cell therapy, TCR gene therapy and immune checkpoint inhibition. In our attempt to explore the immunobiology of naturally-occurring immune responses in patients, we have now elucidated CD137 as a biomarker for bona fide naturally-occurring, tumor-reactive T-cells in cancer and developed a rapid, accurate system to comprehensively isolate tumor infiltrating lymphocytes (TILs) with tumor-rejecting capability directly from resected tumors. CD137+ TILs from various tumor types recognized autologous and HLA-matched tumor in an HLA-restricted manner, resulting in proflammatory cytokine production and cancer killing in vitro, and tumor growth inhibition in vivo; CD137-negative TILs did not. More so, multiparameter phenotyping of the CD137+ TIL subset revealed a vast network of expressed negative immunoregulatory molecules and opportunities for immune checkpoint inhibition. Our findings implicate a role for CD137 in the immunobiology of human cancer where it is preferentially expressed on the tumor-reactive TIL subset, and provides a new roadmap for the rational design of cancer immunotherapy trials. Citation Format: Daniel J. Powell, Jr. Identification and enrichment for rare naturally occurring tumor-reactive T cells in the tumor microenvironment reveals new and diverse opportunities for cancer immunotherapy. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A85.