Steven A. Rosenberg

Identification of HLA class II-restricted T cell receptors against shared PIK3CA mutations in patients with epithelial cancers

PIK3CA is the third most common mutated gene in epithelial cancers, behind only TP53 and KRAS . Somatic PIK3CA mutations occur predominantly in the protein’s hotspot locations H1047, E545, E542 and N345, and evidence of those alterations eliciting immune responses has been limited. HLA class I-restricted TCRs against E545K and H1047L have, only recently, been identified following in vitro sensitization (IVS) with peripheral blood lymphocytes (PBL) from normal donors. In this study, we examined the immunogenicity of PIK3CA in patients with epithelial cancers of diverse histology, by testing TIL and antigen-experienced PBL against autologous hotspot PIK3CA mutations. Two distinct TCRs specific to PIK3CA N345K , restricted by HLA-class II pair DPB1*04:01/DPA1*01:03, were identified in a patient with colon cancer, following independently a TIL screen and an IVS of memory CD4 + T cells from the peripheral blood. Patient PBL engineered to express each TCR suppressed the in vitro growth of two cell lines modified to express the DPB1*04:01/DPA1*01:03 pair and full length PIK3CA N345K protein. A TCR specific to the PIK3CA E545K , restricted by HLA-DRB1*04:01, was also identified following IVS of memory CD4 + PBL from another patient, with rectal cancer. Autologous PBL, gene engineered to express the PIK3CA E545K -specific TCR, suppressed the in vitro growth of two cancer cell lines, which endogenously expressed the PIK3CA E545K neoantigen. This study identified three PIK3CA-specific TCRs against two shared mutations, restricted by common HLA-class II molecules, using antigen-experienced TIL and memory PBL from patients with epithelial cancers and may further allow the development of off-the-shelf T cell immunotherapy strategies targeting PIK3CA.

Selective expansion of T-cell receptor engineered T cells with increased stem-like phenotypes using neoantigen stimulation

Background Adoptive transfer of T-cell receptor-engineered T cells (TCR-T cells) has shown promising efficacy in solid tumor treatment, but achieving clinical benefit typically requires infusion of tens of billions of cells. The commonly used rapid expansion protocol (REP), based on the CD3-agonistic OKT3 antibody and irradiated allogeneic feeder cells, exponentially expands tumor-infiltrating lymphocytes (TILs). However, the effect of REP on TCR-T cell frequency and phenotype remains unclear. This study aimed to evaluate the impact of REP on TCR-T cells and to assess the potential of a neoantigen-specific stimulation platform, NeoExpand, as a strategy to selectively expand TCR-T cells with favorable phenotypes. Methods We compared the effects of REP and NeoExpand on the frequency, yield, and phenotype of TCR-T cells engineered against shared neoantigens. Various autologous antigen-presenting cell (APC) types, including dendritic cells and bulk peripheral blood mononuclear cells (PBMCs), were tested as stimulators in NeoExpand. Additionally, we combined NeoExpand with CD3 activation, with or without allogeneic feeders, to improve scalability. The role of exogenous interleukin (IL)-21 in shaping TCR-T cell phenotypes was also investigated. Results REP impaired the frequency and phenotype of TCR-T cells, particularly CD4 + and CD8 + subsets matched to their engineered TCRs. In contrast, NeoExpand selectively increased TCR-T cell frequency and improved their phenotypes but did not consistently achieve higher total yields compared with REP. Among the tested autologous APCs, PBMCs effectively supported selective expansion. Further optimization using PBMCs as APCs revealed that combining NeoExpand with CD3 activation enabled exponential expansion without compromising selectivity. Inclusion of IL-21 during NeoExpand promoted a naïve/stem-like phenotype in CD8 + TCR-T cells, with minimal effect on CD4 + TCR-T cells. Conclusions These findings demonstrate that NeoExpand enables selective expansion of TCR-T cells while preserving favorable phenotypes and scalability. The approach supports further clinical evaluation of NeoExpand as a strategy to generate high-quality TCR-T cell products for adoptive cell therapy.

Adoptive Cell Transfer of Tumor-Infiltrating Lymphocytes for Metastatic Acral Lentiginous Melanoma

PURPOSE Acral lentiginous melanoma is a subtype of cutaneous melanoma arising from palmar, plantar, or subungual skin. These tumors are characterized by aggressive biology, a low tumor mutational burden (TMB), and diminished sensitivity to immune checkpoint blockade. It is unknown whether adoptive cell transfer of tumor-infiltrating lymphocytes (ACT-TIL) has efficacy in patients with acral melanoma. METHODS We analyzed prospectively collected data from 442 patients with metastatic cutaneous melanoma who were treated on clinical trials of ACT-TIL at a single institution between 1999 and 2018. Although blinded to treatment outcome and genomic data, we retrospectively identified patients who had acral subtype on the basis of clinicopathologic data available at the time of diagnosis. We then evaluated the ACT-TIL treatment outcomes of patients with acral melanoma and compared them with contemporaneously treated patients with nonacral melanoma. RESULTS Out of 442 included patients, 30 (7%) had acral melanoma while 412 (93%) had nonacral melanoma. Cohorts had similar clinical characteristics, protocol enrollment, and treatment-related factors. The objective response rate to ACT-TIL in patients with acral and nonacral melanomas was 43% and 40%, respectively ( P = .87), with 3% and 16% having complete responses (CRs; P = .07). Median progression-free survival was 3.5 and 4.1 months ( P = .40) and median overall survival was 13 and 17 months ( P = .79), respectively. Acral melanomas had lower TMB and ultraviolet mutational signature scores than nonacral melanomas. CONCLUSION ACT-TIL can mediate objective responses in patients with metastatic acral melanoma, and outcomes in patients with acral disease were unexpectedly comparable with those of contemporaneously treated patients with nonacral cutaneous melanoma. Further research is necessary to understand the immunologic basis of responses to ACT-TIL in acral melanoma and to increase the frequency of CRs.

Tumor-infiltrating lymphocyte immunotherapy comes of age: a journey of development in the Surgery Branch, NCI

The early development of tumor-infiltrating lymphocytes into an effective clinical strategy was fundamentally the work of hundreds of scientists and clinicians within the Surgery Branch of the National Cancer Institute under the leadership of Steven Rosenberg. That journey brought new insights into the tumor-immune cell interface and ultimately helped create a new first-in-class therapeutic for patients with metastatic cancer.

Utilization of primary tumor samples for cancer neoantigen discovery

BACKGROUND: The use of tumor-infiltrating T lymphocytes (TIL) that recognize cancer neoantigens has led to lasting remissions in metastatic melanoma and certain cases of metastatic epithelial cancer. For the treatment of the latter, selecting cells for therapy typically involves laborious screening of TIL for recognition of autologous tumor-specific mutations, detected through next-generation sequencing of freshly resected metastatic tumors. Our study explored the feasibility of using archived formalin-fixed, paraffin-embedded (FFPE) primary tumor samples for cancer neoantigen discovery, to potentially expedite this process and reduce the need for resections normally required for tumor sequencing. METHOD: Whole-exome sequencing was conducted on matched primary and metastatic colorectal cancer samples from 22 patients. The distribution of metastatic tumor mutations that were confirmed as neoantigens through cognate TIL screening was evaluated in the corresponding primary tumors. Mutations unique to primary tumors were screened for recognition by metastasis-derived TIL and circulating T lymphocytes. RESULTS: We found that 25 (65.8%) of the 38 validated neoantigens identified in metastatic tumors from 18 patients with colorectal cancer were also present in matched primary tumor samples. This included all 12 neoantigens encoded by putative cancer driver genes, which are generally regarded as superior targets for adoptive cell therapy. The detection rate for other neoantigens, representing mutations without an established role in cancer biology, was 50% (13/26). Gene products encoding neoantigens detected in the primary tumors were not more likely to be clonal or broadly distributed among the analyzed metastatic lesions compared with those undetected in the primary tumors. Additionally, we found that mutations detected only in primary tumor samples did not elicit recognition by metastatic tumor-derived TIL but could elicit specific recognition by the autologous circulating memory T cells. CONCLUSIONS: Our findings indicate that primary FFPE tumor-derived screening libraries could be used to discover most neoantigens present in metastatic tumors requiring treatment. Furthermore, this approach can reveal additional neoantigens not present in resected metastatic tumors, prompting further research to understand their clinical relevance as potential therapeutic targets.

RosenbergScience

Lymphocytes as a living drug for cancer

Using a patient's lymphocytes is approved to treat melanoma and has wider applications.

Neoantigen-specific stimulation of tumor-infiltrating lymphocytes enables effective TCR isolation and expansion while preserving stem-like memory phenotypes

Rapid anti-myeloma activity by T cells expressing an anti-BCMA CAR with a human heavy-chain-only antigen-binding domain

FHVH-T correlated with peak blood FHVH-T levels. Single-cell RNA sequencing revealed a shift toward more differentiated FHVH-T after infusion. Anti-CAR antibody responses were detected in 4 of 12 patients assessed. FHVH-T has powerful, rapid, and durable anti-MM activity.

Using patient-derived tumor organoids from common epithelial cancers to analyze personalized T-cell responses to neoantigens

Adoptive cell transfer of tumor-infiltrating lymphocytes (TIL) can mediate durable complete responses in some patients with common epithelial cancers but does so infrequently. A better understanding of T-cell responses to neoantigens and tumor-related immune evasion mechanisms requires having the autologous tumor as a reagent. We investigated the ability of patient-derived tumor organoids (PDTO) to fulfill this need and evaluated their utility as a tool for selecting T-cells for adoptive cell therapy. PDTO established from metastases from patients with colorectal, breast, pancreatic, bile duct, esophageal, lung, and kidney cancers underwent whole exomic sequencing (WES), to define mutations. Organoids were then evaluated for recognition by autologous TIL or T-cells transduced with cloned T-cell receptors recognizing defined neoantigens. PDTO were also used to identify and clone TCRs from TIL targeting private neoantigens and define those tumor-specific targets. PDTO were successfully established in 38/47 attempts. 75% were available within 2 months, a timeframe compatible with screening TIL for clinical administration. These lines exhibited good genetic fidelity with their parental tumors, especially for mutations with higher clonality. Immunologic recognition assays demonstrated instances of HLA allelic loss not found by pan-HLA immunohistochemistry and in some cases WES of fresh tumor. PDTO could also be used to show differences between TCRs recognizing the same antigen and to find and clone TCRs recognizing private neoantigens. PDTO can detect tumor-specific defects blocking T-cell recognition and may have a role as a selection tool for TCRs and TIL used in adoptive cell therapy.

Cell surface marker-based capture of neoantigen-reactive CD8⁺ T-cell receptors from metastatic tumor digests

Background Cellular immunotherapies using autologous tumor-infiltrating lymphocytes (TIL) can induce durable regression of epithelial cancers in selected patients with treatment-refractory metastatic disease. As the genetic engineering of T cells with tumor-reactive T-cell receptors (TCRs) comes to the forefront of clinical investigation, the rapid, scalable, and cost-effective detection of patient-specific neoantigen-reactive TIL remains a top priority. Methods We analyzed the single-cell transcriptomic states of 31 neoantigen-specific T-cell clonotypes to identify cell surface dysfunction markers that best identified the metastatic transcriptional states enriched with antitumor TIL. We developed an efficient method to capture neoantigen-reactive TCRs directly from resected human tumors based on cell surface co-expression of CD39, programmed cell death protein-1, and TIGIT dysfunction markers (CD8 + TIL TP ). Results TIL TP TCR isolation achieved a high degree of correlation with single-cell transcriptomic signatures that identify neoantigen-reactive TCRs, making it a cost-effective strategy using widely available resources. Reconstruction of additional TIL TP TCRs from tumors identified known and novel antitumor TCRs, showing that at least 39.5% of TIL TP TCRs are neoantigen-reactive or tumor-reactive. Despite their substantial enrichment for neoantigen-reactive TCR clonotypes, clonal dynamics of 24 unique antitumor TIL TP clonotypes from four patients indicated that most in vitro expanded TIL TP populations failed to demonstrate neoantigen reactivity, either by loss of neoantigen-reactive clones during TIL expansion, or through functional impairment during cognate neoantigen recognition. Conclusions While direct usage of in vitro-expanded CD8 + TIL TP as a source for cellular therapy might be precluded by profound TIL dysfunction, isolating TIL TP represents a streamlined effective approach to rapidly identify neoantigen-reactive TCRs to design engineered cellular immunotherapies against cancer.

Specific recognition of an FGFR2 fusion by tumor infiltrating lymphocytes from a patient with metastatic cholangiocarcinoma

Background Metastatic cholangiocarcinoma (CC), a form of gastrointestinal cancer that originates from the bile ducts, cannot be cured by currently available therapies, and is associated with dismal prognosis. In a previous case report, adoptive transfer of autologous tumor infiltrating lymphocytes (TILs), the majority of which recognized a tumor-specific point mutation, led to a profound and durable cancer regression in a patient with metastatic CC. Thus, more effective treatment for patients with this disease may be developed by using TILs that target cancer-specific mutations, but also other genetic aberrations such as gene fusions. In this context, fusions that involve fibroblast growth factor receptor 2 ( FGFR2 ) and function as oncogenes in a subset of patients with intrahepatic CC (ICC) represent particularly attractive targets for adoptive cell therapy. However, no study to date has explored whether FGFR2 fusions can be recognized by patients’ T cells. Method To address whether FGFR2 fusions can be recognized by patients’ T cells, we tested TILs from four patients with FGFR2 fusion-positive ICC for recognition of peptides and minigenes that represented the breakpoint regions of these fusions, which were unique to each of the four patients. Results We found that CD4 + TILs from one patient specifically recognized the breakpoint region of a unique FGFR2-TDRD1 (tudor domain-containing 1) fusion, and we isolated a T-cell receptor responsible for its recognition. Conclusions This finding suggests that FGFR2 fusion-reactive TILs can be isolated from some patients with metastatic ICC, and thus provides a rationale for future exploration of T cell-based therapy targeting FGFR2 fusions in patients with cancer. Furthermore, it augments the rationale for extending such efforts to other types of solid tumors hallmarked by oncogenic gene fusions.

Data from Adoptive Transfer of Autologous Natural Killer Cells Leads to High Levels of Circulating Natural Killer Cells but Does Not Mediate Tumor Regression

Abstract Purpose: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) can mediate regression of metastatic melanoma. However, many patients with cancer are ineligible for such treatment because their TIL do not expand sufficiently or because their tumors have lost expression of antigens and/or MHC molecules. Natural killer (NK) cells are large granular lymphocytes that lyse tumor cells in a non–MHC-restricted manner. Therefore, we initiated in a clinical trial to evaluate the efficacy of adoptively transferred autologous NK cells to treat patients with cancers who were ineligible for treatment with TIL. Experimental Design: Patients with metastatic melanoma or renal cell carcinoma were treated with adoptively transferred in vitro activated autologous NK cells after the patients received a lymphodepleting but nonmyeloablative chemotherapy regimen. Clinical responses and persistence of the adoptively transferred cells were evaluated. Results: Eight patients were treated with an average of 4.7 × 10¹⁰ (± 2.1 × 10¹⁰) NK cells. The infused cells exhibited high levels of lytic activity in vitro. Although no clinical responses were observed, the adoptively transferred NK cells seemed to persist in the peripheral circulation of patients for at least one week posttransfer and, in some patients, for several months. However, the persistent NK cells in the circulation expressed significantly lower levels of the key activating receptor NKG2D and could not lyse tumor cell targets in vitro unless reactivated with IL-2. Conclusions: The persistent NK cells could mediate antibody-dependent cell-mediated cytotoxicity without cytokine reactivation in vitro, which suggests that coupling adoptive NK cell transfer with monoclonal antibody administration deserves evaluation. Clin Cancer Res; 17(19); 6287–97.

Data from Adoptive Transfer of Autologous Natural Killer Cells Leads to High Levels of Circulating Natural Killer Cells but Does Not Mediate Tumor Regression

Abstract Purpose: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) can mediate regression of metastatic melanoma. However, many patients with cancer are ineligible for such treatment because their TIL do not expand sufficiently or because their tumors have lost expression of antigens and/or MHC molecules. Natural killer (NK) cells are large granular lymphocytes that lyse tumor cells in a non–MHC-restricted manner. Therefore, we initiated in a clinical trial to evaluate the efficacy of adoptively transferred autologous NK cells to treat patients with cancers who were ineligible for treatment with TIL. Experimental Design: Patients with metastatic melanoma or renal cell carcinoma were treated with adoptively transferred in vitro activated autologous NK cells after the patients received a lymphodepleting but nonmyeloablative chemotherapy regimen. Clinical responses and persistence of the adoptively transferred cells were evaluated. Results: Eight patients were treated with an average of 4.7 × 10¹⁰ (± 2.1 × 10¹⁰) NK cells. The infused cells exhibited high levels of lytic activity in vitro. Although no clinical responses were observed, the adoptively transferred NK cells seemed to persist in the peripheral circulation of patients for at least one week posttransfer and, in some patients, for several months. However, the persistent NK cells in the circulation expressed significantly lower levels of the key activating receptor NKG2D and could not lyse tumor cell targets in vitro unless reactivated with IL-2. Conclusions: The persistent NK cells could mediate antibody-dependent cell-mediated cytotoxicity without cytokine reactivation in vitro, which suggests that coupling adoptive NK cell transfer with monoclonal antibody administration deserves evaluation. Clin Cancer Res; 17(19); 6287–97.

Rosenberg

Data from Durable Complete Responses in Heavily Pretreated Patients with Metastatic Melanoma Using T-Cell Transfer Immunotherapy

Abstract Purpose: Most treatments for patients with metastatic melanoma have a low rate of complete regression and thus overall survival in these patients is poor. We investigated the ability of adoptive cell transfer utilizing autologous tumor-infiltrating lymphocytes (TIL) to mediate durable complete regressions in heavily pretreated patients with metastatic melanoma. Experimental Design: Ninety-three patients with measurable metastatic melanoma were treated with the adoptive transfer of autologous TILs administered in conjunction with interleukin-2 following a lymphodepleting preparative regimen on three sequential clinical trials. Ninety-five percent of these patients had progressive disease following a prior systemic treatment. Median potential follow-up was 62 months. Results: Objective response rates by Response Evaluation Criteria in Solid Tumors (RECIST) in the 3 trials using lymphodepleting preparative regimens (chemotherapy alone or with 2 or 12 Gy irradiation) were 49%, 52%, and 72%, respectively. Twenty of the 93 patients (22%) achieved a complete tumor regression, and 19 have ongoing complete regressions beyond 3 years. The actuarial 3- and 5-year survival rates for the entire group were 36% and 29%, respectively, but for the 20 complete responders were 100% and 93%. The likelihood of achieving a complete response was similar regardless of prior therapy. Factors associated with objective response included longer telomeres of the infused cells, the number of CD8⁺CD27⁺ cells infused, and the persistence of the infused cells in the circulation at 1 month (all P₂ < 0.001). Conclusions: Cell transfer therapy with autologous TILs can mediate durable complete responses in patients with metastatic melanoma and has similar efficacy irrespective of prior treatment. Clin Cancer Res; 17(13); 4550–7.

Data from CD8+ Enriched “Young” Tumor Infiltrating Lymphocytes Can Mediate Regression of Metastatic Melanoma

Abstract Purpose: Tumor‐infiltrating lymphocytes (TIL) and interleukin (IL)-2 administered following lymphodepletion can cause the durable complete regression of bulky metastatic melanoma in patients refractory to approved treatments. However, the generation of a unique tumor-reactive TIL culture for each patient may be prohibitively difficult. We therefore investigated the clinical and immunologic impact of unscreened, CD8+ enriched “young” TIL. Experimental Design: Methods were developed for generating TIL that minimized the time in culture and eliminated the individualized tumor-reactivity screening step. Thirty-three patients were treated with these CD8+ enriched young TIL and IL-2 following nonmyeloablative lymphodepletion (NMA). Twenty-three additional patients were treated with CD8+ enriched young TIL and IL-2 after lymphodepletion with NMA and 6 Gy of total body irradiation. Results: Young TIL cultures for therapy were successfully established from 83% of 122 consecutive melanoma patients. Nineteen of 33 patients (58%) treated with CD8+ enriched young TIL and NMA had an objective response (Response Evaluation Criteria in Solid Tumors) including 3 complete responders. Eleven of 23 patients (48%) treated with TIL and 6 Gy total body irradiation had an objective response including 2 complete responders. At 1 month after TIL infusion the absolute CD8+ cell numbers in the periphery were highly correlated with response. Conclusions: This study shows that a rapid and simplified method can be used to reliably generate CD8+ enriched young TIL for administration as an individualized therapy for advanced melanoma, and may allow this potentially effective treatment to be applied at other institutions and to reach additional patients. Clin Cancer Res; 16(24); 6122–31.

Data from CD8+ Enriched “Young” Tumor Infiltrating Lymphocytes Can Mediate Regression of Metastatic Melanoma

Abstract Purpose: Tumor‐infiltrating lymphocytes (TIL) and interleukin (IL)-2 administered following lymphodepletion can cause the durable complete regression of bulky metastatic melanoma in patients refractory to approved treatments. However, the generation of a unique tumor-reactive TIL culture for each patient may be prohibitively difficult. We therefore investigated the clinical and immunologic impact of unscreened, CD8+ enriched “young” TIL. Experimental Design: Methods were developed for generating TIL that minimized the time in culture and eliminated the individualized tumor-reactivity screening step. Thirty-three patients were treated with these CD8+ enriched young TIL and IL-2 following nonmyeloablative lymphodepletion (NMA). Twenty-three additional patients were treated with CD8+ enriched young TIL and IL-2 after lymphodepletion with NMA and 6 Gy of total body irradiation. Results: Young TIL cultures for therapy were successfully established from 83% of 122 consecutive melanoma patients. Nineteen of 33 patients (58%) treated with CD8+ enriched young TIL and NMA had an objective response (Response Evaluation Criteria in Solid Tumors) including 3 complete responders. Eleven of 23 patients (48%) treated with TIL and 6 Gy total body irradiation had an objective response including 2 complete responders. At 1 month after TIL infusion the absolute CD8+ cell numbers in the periphery were highly correlated with response. Conclusions: This study shows that a rapid and simplified method can be used to reliably generate CD8+ enriched young TIL for administration as an individualized therapy for advanced melanoma, and may allow this potentially effective treatment to be applied at other institutions and to reach additional patients. Clin Cancer Res; 16(24); 6122–31.

Data from Determinants of Successful CD8⁺ T-Cell Adoptive Immunotherapy for Large Established Tumors in Mice

Abstract Purpose: Adoptive cell transfer (ACT) of tumor infiltrating or genetically engineered T cells can cause durable responses in patients with metastatic cancer. Multiple clinically modifiable parameters can comprise this therapy, including cell dose and phenotype, in vivo antigen restimulation, and common gamma-chain (γc) cytokine support. However, the relative contributions of each these individual components to the magnitude of the antitumor response have yet to be quantified. Experimental Design: To systematically and quantitatively appraise each of these variables, we employed the Pmel-1 mouse model treating large, established B16 melanoma tumors. In addition to cell dose and magnitude of in vivo antigen restimulation, we also evaluated the relative efficacy of central memory (TCM), effector memory (TEM), and stem cell memory (TSCM) subsets on the strength of tumor regression as well as the dose and type of clinically available γc cytokines, including IL-2, IL-7, IL-15, and IL-21. Results: We found that cell dose, T-cell differentiation status, and viral vaccine titer each were correlated strongly and significantly with the magnitude of tumor regression. Surprisingly, although the total number of IL-2 doses was correlated with tumor regression, no significant benefit to prolonged (≥6 doses) administration was observed. Moreover, the specific type and dose of γc cytokine only moderately correlated with response. Conclusion: Collectively, these findings elucidate some of the key determinants of successful ACT immunotherapy for the treatment of cancer in mice and further show that γc cytokines offer a similar ability to effectively drive antitumor T-cell function in vivo. Clin Cancer Res; 17(16); 5343–52.

Data from Development of Human Anti-Murine T-Cell Receptor Antibodies in Both Responding and Nonresponding Patients Enrolled in TCR Gene Therapy Trials

Abstract Purpose: Immune responses to gene-modified cells are a concern in the field of human gene therapy, as they may impede effective treatment. We conducted 2 clinical trials in which cancer patients were treated with lymphocytes genetically engineered to express murine T-cell receptors (mTCR) specific for tumor-associated antigens p53 and gp100. Experimental Design: Twenty-six patients treated with autologous lymphocytes expressing mTCR had blood and serum samples available for analysis. Patient sera were assayed for the development of a humoral immune response. Adoptive cell transfer characteristics were analyzed to identify correlates to immune response. Results: Six of 26 (23%) patients' posttreatment sera exhibited specific binding of human anti-mTCR antibodies to lymphocytes transduced with the mTCR. Antibody development was found in both responding and nonresponding patients. The posttreatment sera of 3 of these 6 patients mediated a 60% to 99% inhibition of mTCR activity as measured by a reduction in antigen-specific interferon-γ release. Detailed analysis of posttreatment serum revealed that antibody binding was β-chain specific in 1 patient whereas it was α-chain specific in another. Conclusions: A subset of patients treated with mTCR-engineered T cells developed antibodies directed to the mTCR variable regions and not to the constant region domains common to all mTCR. Overall, the development of a host immune response was not associated with the level of transduced cell persistence or response to therapy. In summary, patients treated with mTCR can develop an immune response to gene-modified cells in a minority of cases, but this may not affect clinical outcome. Clin Cancer Res; 16(23); 5852–61.

Data from Development of Human Anti-Murine T-Cell Receptor Antibodies in Both Responding and Nonresponding Patients Enrolled in TCR Gene Therapy Trials

Abstract Purpose: Immune responses to gene-modified cells are a concern in the field of human gene therapy, as they may impede effective treatment. We conducted 2 clinical trials in which cancer patients were treated with lymphocytes genetically engineered to express murine T-cell receptors (mTCR) specific for tumor-associated antigens p53 and gp100. Experimental Design: Twenty-six patients treated with autologous lymphocytes expressing mTCR had blood and serum samples available for analysis. Patient sera were assayed for the development of a humoral immune response. Adoptive cell transfer characteristics were analyzed to identify correlates to immune response. Results: Six of 26 (23%) patients' posttreatment sera exhibited specific binding of human anti-mTCR antibodies to lymphocytes transduced with the mTCR. Antibody development was found in both responding and nonresponding patients. The posttreatment sera of 3 of these 6 patients mediated a 60% to 99% inhibition of mTCR activity as measured by a reduction in antigen-specific interferon-γ release. Detailed analysis of posttreatment serum revealed that antibody binding was β-chain specific in 1 patient whereas it was α-chain specific in another. Conclusions: A subset of patients treated with mTCR-engineered T cells developed antibodies directed to the mTCR variable regions and not to the constant region domains common to all mTCR. Overall, the development of a host immune response was not associated with the level of transduced cell persistence or response to therapy. In summary, patients treated with mTCR can develop an immune response to gene-modified cells in a minority of cases, but this may not affect clinical outcome. Clin Cancer Res; 16(23); 5852–61.

Supplementary Data from CD8+ Enriched “Young” Tumor Infiltrating Lymphocytes Can Mediate Regression of Metastatic Melanoma

Supplementary Figure S1; Supplementary Table S1.