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10.1172/JCI175790
1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
Find articles by Bear, A. in: JCI | PubMed | Google Scholar
1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
Find articles by Nadler, R. in: JCI | PubMed | Google Scholar
1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
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1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
Find articles by Stanton, K. in: JCI | PubMed | Google Scholar
1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
Find articles by Xu, C. in: JCI | PubMed | Google Scholar
1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
Find articles by Saporito, R. in: JCI | PubMed | Google Scholar
1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
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1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
Find articles by Baroja, M. in: JCI | PubMed | Google Scholar
1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
Find articles by Blanchard, T. in: JCI | PubMed | Google Scholar
1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
Find articles by Elliott, M. in: JCI | PubMed | Google Scholar
1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
Find articles by Ford, M. in: JCI | PubMed | Google Scholar
1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
Find articles by Jones, R. in: JCI | PubMed | Google Scholar
1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
Find articles by Patel, S. in: JCI | PubMed | Google Scholar
1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
Find articles by Brennan, A. in: JCI | PubMed | Google Scholar
1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
Find articles by O’Neil, Z. in: JCI | PubMed | Google Scholar
1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
Find articles by Powell, D. in: JCI | PubMed | Google Scholar
1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
Find articles by Vonderheide, R. in: JCI | PubMed | Google Scholar
1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
Find articles by Linette, G. in: JCI | PubMed | Google Scholar
1Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine,
2The College of Arts and Sciences,
3Abramson Cancer Center, and
4Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
5MSBioworks, Ann Arbor, Michigan, USA.
6Department of Pathology and Laboratory Medicine and
7Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Address correspondence to: Beatriz M. Carreno, 3400 Civic Center Blvd, Rm8-103, Philadelphia, Pennsylvania, 19104-5157, USA. Phone: 215.573.7044; Email: bcarreno@upenn.edu.
Authorship note: RBN and MHO contributed equally to this work.
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Authorship note: RBN and MHO contributed equally to this work.
Published September 17, 2024 - More info
Published in Volume 134, Issue 21 on November 1, 2024
Related article:
Abstract
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.
Authors
× AbstractBACKGROUND. Neoantigens derived from KRASMUT have been described, but the fine antigen specificity of T cell responses directed against these epitopes is poorly understood. Here, we explore KRASMUT immunogenicity and the properties of 4 T cell receptors (TCRs) specific for KRASG12V restricted to the HLA-A3 superfamily of class I alleles.
METHODS. A phase 1 clinical vaccine trial targeting KRASMUT was conducted. TCRs targeting KRASG12V restricted to HLA-A*03:01 or HLA-A*11:01 were isolated from vaccinated patients or healthy individuals. A comprehensive analysis of TCR antigen specificity, affinity, crossreactivity, and CD8 coreceptor dependence was performed. TCR lytic activity was evaluated, and target antigen density was determined by quantitative immunopeptidomics.
RESULTS. Vaccination against KRASMUT resulted in the priming of CD8+ and CD4+ T cell responses. KRASG12V -specific natural (not affinity enhanced) TCRs exhibited exquisite specificity to mutated protein with no discernible reactivity against KRASWT. TCR-recognition motifs were determined and used to identify and exclude crossreactivity to noncognate peptides derived from the human proteome. Both HLA-A*03:01 and HLA-A*11:01–restricted TCR-redirected CD8+ T cells exhibited potent lytic activity against KRASG12V cancers, while only HLA-A*11:01–restricted TCR-T CD4+ T cells exhibited antitumor effector functions consistent with partial coreceptor dependence. All KRASG12V-specific TCRs displayed high sensitivity for antigen as demonstrated by their ability to eliminate tumor cell lines expressing low levels of peptide/HLA (4.4 to 242) complexes per cell.
CONCLUSION. This study identifies KRASG12V-specific TCRs with high therapeutic potential for the development of TCR-T cell therapies.
TRIAL REGISTRATION. ClinicalTrials.gov NCT03592888.
FUNDING. AACR SU2C/Lustgarten Foundation, Parker Institute for Cancer Immunotherapy, and NIH.
IntroductionT cell recognition of cancer antigens represents the end effector mechanism of successful cancer immunotherapy (1). Advances in the areas of T cell biology, gene engineering, and antigen identification have nurtured strategies to redirect T cell antigen specificity against cancer cells. Indeed, CAR-T cell therapy for the treatment of hematological malignancies is now widely available (2). Adoptive cell therapy strategies utilizing T cells redirected with tumor-specific T cell receptors (TCRs) (TCR-T) have demonstrated promising clinical results in subsets of cancer patients (3–5); however, fundamental challenges exist, including antigen identification.
Neoantigens arising from recurrent activating mutations in oncogenic driver genes are attractive immunotherapeutic targets due to limited clonal heterogeneity and treatment generalizability across patients and tumor types (6). To date, shared neoantigens of mutant TP53, PIKC3A, and KRAS among others have been described (7). KRAS mutations are observed in up to 20% of all human cancers and drive tumorigenesis in the 3 most lethal cancers in the United States, including adenocarcinomas of the pancreas (PAAD: 80%–90%), colon (COAD: 40%–50%), and lung (LUAD: 30%–40%) (8). The vast majority of KRAS mutations in these tumors occur at the codon 12 position (9), leading to hyperactivation of MAPK and PI3K-AKT downstream effector signaling pathways (10). Among these tumor types, amino acid substitutions at codon position 12 most often involve glycine (G) to aspartic acid (D), valine (V), cysteine (C), or arginine (R) transitions. While G12C and G12R mutations are preferentially observed among LUAD and PAAD tumors, respectively, G12D and G12V mutations remain highly prevalent across the 3 tumor types. Clinical case reports suggest mutant KRAS (KRASMUT) may be amenable to targeting by TCR-based therapies in select patients with KRASG12D tumors who are HLA-C*08:02+ (11, 12). Immunopeptidomic studies performed by our group and others highlight the HLA-A3 superfamily of class I alleles (A*03:01, A*11:01, A*31:01, A*33:01, and others), which share overlapping peptide repertoires (13, 14), as capable of processing and presenting nomaner and decamer epitopes of KRASWT and KRASMUT spanning amino acid residues 8–16 (VVGAXGVGK) and 7–16 (VVVGAXGVGK), respectively, with X signifying the amino acid at codon position 12 (15–17).
The CD8 coreceptor functions to enhance TCR avidity through stabilization of the TCR:peptide/HLA (pHLA) immune synapse via binding to the α3 domain of HLA-I molecules (18). An inherent limitation of TCR-T therapy using HLA-I–restricted TCRs is an inability to leverage CD4+ T cell immunity. CD4+ T cells may be directly cytotoxic and promote antigen-specific help during several phases of the immune response that improves the in vivo persistence and antitumor activity of tumor-specific CD8+ T cells (19). Transgenic expression of CD8αβ may enhance the antitumor activity of CD4+ T cells redirected with HLA-I–restricted TCRs, but such a strategy requires further engineering of T cells. Select TCRs of high pHLA avidity function independently of CD8 coreceptor interactions, allowing transgenic TCR expression on CD4+ T cells to recruit their effector functions (20, 21).
Here, we report a phase 1 clinical trial of autologous mature dendritic cell (mDC) vaccination targeting KRASMUT (mDC3/8-KRAS). Vaccination resulted in the priming of T cell immunity against KRASMUT in select subjects, including CD8+ T cell immunity against KRASG12V in an HLA-A*11:01+ patient. We further explored KRASG12V restricted to HLA-A*03:01 and HLA-A*11:01 as immunological targets using a panel of 4 TCRs derived from this vaccinated cancer patient and healthy donors. TCRs were highly specific to KRASG12V without crossreactivity to predicted peptides encoded in the human proteome and displayed various degrees of CD8 coreceptor dependence. HLA-A*11:01–restricted TCR-engineered CD8+ and CD4+ T cells exhibited lytic activity against KRASG12V+ tumor cell lines with low-abundance neoantigen expression. These results validate G12V/A*03:01 and G12V/A*11:01 as shared neoantigen targets, which underlies the development of adoptive TCR-T cell therapies for the treatment of KRASMUT cancers.
ResultsVaccination primes KRASMUT-specific T cell responses in pancreatic cancer patients. We conducted an investigator-initiated, phase 1 clinical trial to study the immunogenicity of KRASMUT in PAAD patients (ClinicalTrials.gov NCT03592888) utilizing a previously described autologous, mDC-based (mDC3/8) platform (Figure 1A) (22). Key enrollment criteria included (a) a history of resected PAAD without radiographic or biochemical evidence of disease, (b) the presence of a tumor KRASG12 mutation determined by tumor DNA sequencing, and (c) patient expression of at least 1 HLA-I allele corresponding to previously reported KRASMUT neoantigens (15–17). From July 19, 2018, to April 17, 2024, we enrolled 29 subjects of which 9 subjects received vaccination (Figure 1B). All vaccinated subjects had clinical characteristics typical of patients with resected PAAD (Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/JCI175790DS1). All 9 subjects were vaccinated against 1 or more distinct short (nonamer or decamer) KRASMUT peptides targeting patient-specific HLA-I alleles, and 5 of the 9 patients were also vaccinated against 1 or more distinct long KRASMUT peptides in order to, presumably, target HLA-II alleles (Figure 1B and Supplemental Table 2). The number of peptides administered to each patient ranged from 1 to 7 peptides representing KRASMUT variant present in the patient’s tumor as well as others (Supplemental Table 3).
Figure 1mDC3/8-KRAS vaccination primes KRASMUT-specific T cell immunity in PAAD patients. (A) Trial design. (B) Consolidated standards of reporting trials diagram. (C) Number of vaccine KRASMUT neoantigens per patient that induced IFN-γ+ T cells in ex vivo–expanded PBMCs collected after vaccine priming. (D) Normalized IFN-γ+ ELISpot counts for vaccine KRASMUT neoantigens after priming detected in ex vivo–expanded PBMCs. Spot counts of the nonstimulated controls were subtracted. Responses to short peptides (HLA-I) are indicated in red, and responses to long peptides (HLA-II) are indicated in blue. Symbol shape indicates specific KRASMUT as per legend. (E) Assessment of subject no. 2 HLA-I–restricted T cell responses against 8–16V (blue) and 7–16V (red) peptides by IFN-γ ELISpot assay following ex vivo expansion of week 2 postvaccine PBMCs. Free peptide supplemented to media bound by HLA-I expressed on donor white blood cells (HLA-A*11:01 and -A*03:01) and presented to responding T cells. Monoallelic K562 cells expressing HLA-A*03:01 (APC-A3) or HLA-A*11:01 (APC-A11) were used to identify HLA-I restriction. WT indicates WT KRAS peptide. MUT indicates mutant KRAS peptide. (F) pHLA multimer analysis to assess CD8+ T cell response against 8–16V/A*11:01 and 7–16V/A*11:01 following in vitro expansion of pre- (week –1) and postvaccine (week 10) CD8+ T cells. Successful priming of CD8+ T cell responses to gp10017–25/A*03:01 and NY-ESO60–72/B*07:02 served as positive vaccination controls. (G) Circos plot analysis following TCR-αβ RNA sequencing of FACS-sorted CD8+/multimer+ (8–16V/A*11:01) cells. Statistical differences between groups calculated using Students’ unpaired t test.
All vaccinated subjects received a total of 2 vaccine doses (prime and boost) i.v. Two subjects were vaccinated below the prespecified prime or boost dose levels of 10–30 × 106 and 7–15 × 106 DCs/peptide, respectively (Supplemental Figure 1A). Effective DC maturation using CD40L/IFN-γ, poly I:C, and R848 was confirmed in all subjects via the induction of IL-12p70 production (Supplemental Figure 1B). Vaccination was safe and well tolerated (primary end points) with no subjects experiencing grade 3 or higher adverse events (AEs). The most common AEs observed following vaccination included chills, fatigue, and headache (Supplemental Figure 1C).
KRASMUT-specific T cell responses primed by vaccination (secondary endpoint) were assessed by IFN-γ ELISpot assay following in vitro T cell expansion. Six out of 9 (67%) subjects generated KRASMUT-specific T cell responses following vaccination (Figure 1C). In a subset of subjects, T cell responses were directed at more than one KRASMUT peptide. Two (subjects nos. 2 and 12) out of 9 subjects generated T cell responses to HLA-I–restricted short peptides, whereas the inclusion of long KRASMUT peptides in the vaccine formulation enhanced the immune response rate with 5/5 subjects exhibiting measurable immunity to at least 1 long peptide (Figure 1D). Whether these long peptides elicited CD4 and/or CD8 T cell responses is still being characterized. At a median time to follow-up of 25.3 months, 5 subjects were alive without evidence of tumor recurrence, while 4 subjects had experienced tumor recurrence and died due to disease progression (Supplemental Figure 1D).
Subject 2 was vaccinated against nonamer 8–16V and decamer 7–16V KRASMUT peptides targeting patient HLA-I alleles A*03:01 and A*11:01 along with control peptides gp100 (gp17–25) restricted to HLA-A*03:01 and NY-ESO-1 (NY60–72) restricted to HLA-B*07:02. Analysis of ex vivo–expanded PBMC samples collected at week 2 after vaccination demonstrated a positive immune response against both 8–16V and 7–16V with no reactivity to KRASWT peptides (Figure 1E). To identify the HLA-I–restricting allele of this response, K562 cells (HLA-I negative) were engineered to express a single-chain dimer construct encoding β2-microglobulin linked to either HLA-A*03:01 (K562A*03:01) or HLA-A*11:01 (K562A*11:01) heavy chain (Supplemental Figure 1E) (23). IFN-γ production by ex vivo–expanded PBMC samples was detected in the presence of 8–16V or 7–16V peptide-pulsed K562A*11:01 but not K562A*03:01 cells, indicating this response to be restricted by HLA-A*11:01 and not HLA-A*03:01. pHLA multimer analysis of ex vivo–expanded CD8+ T cells collected before (week –1) and after vaccination (week 10) confirmed de novo priming of a 8–16V/A*11:01–specific CD8+ T cell response and a weaker priming of a 7–16V/A*11:01 response (Figure 1F). No HLA-A*03:01–restricted T cell response against KRASG12V was observed despite successful vaccine-induced priming of CD8+ T cell responses against gp17–25/A*03:01. In subject 24, vaccination against 8–16V and 7–16V KRASMUT peptides targeted to HLA-A*03:01 did not elicit an immune response; however, this patient did not exhibit a response against control gp17–25 peptide, suggesting impaired CD8+ T cell immunity (Supplemental Figure 1F). Subject 12 demonstrated a positive CD8+ T cell response against KRASG12R peptide 3–12R by IFN-γ ELISpot and pHLA multimer assays (Supplemental Figure 1, G and H), a candidate KRASMUT neoantigen with predicted high-binding affinity to HLA-A*33:01 (affinity = 10.5 nM, NetMHC4.0). Notably, no HLA-A*02:01– or HLA-C*08:02–restricted KRASMUT peptide-specific immune responses were observed in vaccinated subjects exhibiting these HLA-I alleles (Supplemental Table 3).
Identification of KRASG12V-specific TCRs in healthy donors and vaccinated patients. In prior work, we utilized a multiomic approach to identify HLA-I–restricted neoantigens derived from KRASMUT (15). We performed biochemical studies to measure pHLA-binding affinity and complex stability, both parameters that correlate with peptide immunogenicity. These studies highlight epitopes of KRASG12V (8–16V and 7–16V) restricted to HLA-A*03:01 and HLA-A*11:01 as exhibiting optimal immunogenic properties relative to other KRASMUT epitopes (Supplemental Figure 1I). In select healthy donors, in vitro priming and T cell expansion assays yielded CD8+ T cell responses against the 7–16V epitope restricted to HLA-A*03:01 and HLA-A*11:01, but not the 8–16V peptide (Supplemental Figure 1J). From these donors, we isolated TCR-αβ pairs specific for 7–16V/A*03:01 (designated as A3V) and 7–16V/A*11:01 (designated as A11Va, A11Vb) (Table 1). Additionally, a TCR-αβ pair specific for 8–16V/A*11:01 (designated as A11Vc) was isolated from an oligoclonal population identified in vaccine subject 2 (Figure 1G and Table 1). Both A3V and A11Va have been previously reported (15), while A11Vb and A11Vc are first introduced here.
Table 1Summary of HLA-A3 superfamily-restricted TCRs specific for KRASG12V
TCRs are highly specific for KRASG12V and recognize distinct peptide-binding motifs. We utilized JASP90_CD8+ reporter cells, which comprise TCR-αβnull Jurkat E6.1 cells engineered to express the CD8αβ coreceptor and a Uni-Vect reporter construct consisting of an nuclear factor of activated T cells–inducible (NFAT-inducible) EGFP reporter to readout TCR signaling (23). JASP90_CD8+ cells were further engineered to express KRASMUT TCR constructs via lentiviral transduction and positively sorted to purity based on CD3 expression (Supplemental Figure 2A). TCR-engineered JASP90_CD8+ cells were cocultured with HLA-I–matched K562 cells pulsed with either KRASWT or KRASG12V synthetic peptides and assessed for TCR activation (EGFP expression) 16 hours later. All TCRs demonstrated specific reactivity to cognate KRASG12V peptides without crossreactivity to KRASWT (Figure 2A). A3V, A11Va, and A11Vb were exclusively reactive against 7–16V, whereas A11Vc exhibited reactivity to 8–16V and 7–16V peptides (Figure 2B).
Figure 2TCRs are specific for KRASG12V and exhibit distinct peptide-binding motifs with crossreactivity to KRASG12C. (A) FACS profiles of TCR-engineered JASP90_CD8+ cells following 16 hours of coculture with HLA-I –matched K562 cells pulsed with KRASWT (black) or cognate KRASG12V (colored) peptide. (B) Bar graphs representing NFAT activation (specific activity, %) of JASP90_CD8+ cells following 16 hours of coculture with HLA-I–matched K562 cells pulsed with 9-mer and 10-mer KRASWT or KRASG12V peptides. (C) Peptide-binding motifs determined by X-scan analysis of TCR A3V, A11Va, A11Vb, and A11Vc depicted as heatmaps (top) and Seq2Logo plots (bottom) using JASP90_CD8+ reporter cells cocultured with HLA-I–matched K562 cells pulsed with positional peptide scanning library peptides. Heatmaps: specific activity = (GFPExp - GFPMin) / (GFPMax – GFPMin); GFPMin = unstimulated, GFPMax = PMA-I. Seq2Logo plots: height of amino acid at each position corresponds to EGFP expression relative to unstimulated and PMA-I conditions. (D) Cell-reporter assay using TCR-engineered JASP90_CD8+ cocultured with K562A*11:01 cells pulsed with titrated levels of cognate G12V versus G12C peptides. Differences in TCR functional avidities for each peptide are displayed as Δlog10(EC50) values. Data are representative of 2 or more experiments.
To define peptide residues critical for TCR engagement (recognition motif), we initially employed an Ala/Gly peptide library (Supplemental Data Set 1) for presentation by HLA-I–matched K562 cells and cocultured with TCR-engineered JASP90_CD8+ cells. Ala/Gly scanning assays identified both anchor (peptide position 2 [P2]) and nonanchor (P4–P8) residues within the 7–16V peptide critical for the activation of each TCR (Supplemental Figure 2B). TCR recognition motif and crossreactivity characterization were explored further by employing a positional peptide library, X-scan (Figure 2C) (24, 25). The X-scan library consisted of 190 synthetic peptides in which each amino acid residue in the 7–16V peptide sequence was substituted by all 19 remaining l-amino acids (Supplemental Data Set 2). As expected, limited amino acid substitutions were tolerated at residues P2/3 and P10, corresponding to N- and C-terminal anchor positions, respectively. Notably, A11Vc functioned independently of all amino acid substitutions at P1 of the 10-mer peptide while A11Va and A11Vb recognition was affected by select P1 amino acid substitutions. For all TCRs, limited substitutions were tolerated for valine at P6 corresponding to the codon 12 mutant position. A11Va-c demonstrated crossreactivity to KRASG12C (7–16C), which is the most prevalent KRAS mutation observed in human LUAD. We compared the functional avidities of A11Va-c against cognate KRASG12V versus KRAS
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