Annamalai Selvakumar

Survey of naturally occurring CD4+ T cell responses against NY-ESO-1 in cancer patients: Correlation with antibody responses

NY-ESO-1 is one of the most immunogenic proteins described in human cancers, based on its capacity to elicit simultaneous antibody and CD8+ T cell responses in vivo. Although HLA class II restricted epitopes from NY-ESO-1 have been identified, no broad survey has yet established the status of natural CD4+ T cell responses in cancer patients in relation to CD8+ and antibody responses. We used a recently developed general strategy for monitoring CD4+ responses that overcomes the need for prior knowledge of epitope or HLA restriction to analyze a series of 31 cancer patients and healthy donors for the presence of CD4+ T cells to NY-ESO-1, and related this response to NY-ESO-1 expression in tumor cells and serum antibodies to NY-ESO-1. None of the 18 patients that tested seronegative for NY-ESO-1 had detectable CD4+ T cell responses. On the contrary, 11 of 13 cancer patients with serum antibodies to NY-ESO-1 had polyclonal CD4+ T cell responses directed against various known and previously undescribed NY-ESO-1 epitopes. NY-ESO-1 peptide 80–109 was the most immunogenic, with 10 of 11 patients responding to this peptide. We show here that 12-mer determinants from NY-ESO-1 eliciting a CD4+ T cell response were peptide 87–98 with promiscuous HLA class II presentation, peptide 108–119 restricted by HLA-DP4, and peptides 121–132 and 145–156, both shorter epitopes from previously described HLA-DR4 peptides, also presented by HLA-DR7. This study represents the next step in compiling a comprehensive picture of the adaptive immune response to NY-ESO-1, and provides a general strategy for analyzing the CD4+ T cell response to other tumor antigens eliciting a humoral immune response.

Vaccine-Induced CD4+ T Cell Responses to MAGE-3 Protein in Lung Cancer Patients

MAGE-3 is the most commonly expressed cancer testis Ag and thus represents a prime target for cancer vaccines, despite infrequent natural occurrence of MAGE-3-specific immune responses in vivo. We report in this study the successful induction of Ab, CD8+, and CD4+ T cells in nonsmall cell lung cancer patients vaccinated with MAGE-3 recombinant protein. Two cohorts were analyzed: one receiving MAGE-3 protein alone, and one receiving MAGE-3 protein with adjuvant AS02B. Of nine patients in the first cohort, three developed marginal Ab titers and another one had a CD8+ T cell response to HLA-A2-restricted peptide MAGE-3 271–279. In contrast, of eight patients from the second cohort vaccinated with MAGE-3 protein and adjuvant, seven developed high-titered Abs to MAGE-3, and four had a strong concomitant CD4+ T cell response to HLA-DP4-restricted peptide 243–258. One patient simultaneously developed CD8+ T cells to HLA-A1-restricted peptide 168–176. The novel monitoring methodology used in this MAGE-3 study establishes that protein vaccination induces clear CD4+ T cell responses that correlate with Ab production. This development provides the framework for further evaluating integrated immune responses in vaccine settings and for optimizing these responses for clinical benefit.

KIR2DS1-Positive NK Cells Mediate Alloresponse against the C2 HLA-KIR Ligand Group In Vitro

The inhibitory 2DL1 and activating 2DS1 killer Ig-like receptors (KIR) both have shared ligand specificity for codon sequences in the C2 group HLA-Cw Ags. In this study, we have investigated NK cell activation by allogeneic target cells expressing different combinations of the HLA-KIR ligand groups C1, C2, and Bw4. We demonstrate that fresh NK cells as well as IL-2-propagated NK cells from 2DS1-positive donors that are homozygous for the C1 ligand group are activated in vitro by B lymphoblastoid cell lines expressing the C2 group. This response is, in part, due to the absence of C1 group recognition mediated by the inhibitory receptor 2DL2/3. This “missing self” alloresponse to C2, however, is rarely observed in NK cells from donors lacking 2DS1. Even in presence of 2DS1, the NK alloresponse is dramatically reduced in donors that have C2 group as “self.” Analysis of selected NK clones that express 2DS1 mRNA and lack mRNA for 2DL1 demonstrates that activation by the C2 ligand and mAb cross-linking of 2DS1 in these clones induces IFN-γ. Furthermore, this C2 group-induced activation is inhibited by Abs to both HLA class I and the receptor. Collectively, these studies demonstrate that NK cells from 2DS1-positive donors are activated by target cells that express the C2 group as an alloantigen. This leads to increased IFN-γ-positive fresh NK cells and induces NK allocytotoxicity in IL2-propagated polyclonal NK cells and NK clones. This study also provides support for the concept that incompatibility for the HLA-KIR ligand groups C1, C2, and Bw4 dominates NK alloactivation in vitro.

Polymorphism and domain variability of human killer cell inhibitory receptors

Summary: Thirty‐two different full‐length cDNA clones for human killer cell inhibitory receptors (KIR) have been identified. They all belong to the immunoglobulin superfamily and encode mature proteins with one, two or three extracellular Ig domains. The inhibitory receptors have nearly identical transmembrane domains and cytoplasmic domains ranging in length from 76 to 95 amino‐acid residues. The activating receptors have a characteristic transmembrane domain with a charged lysine residue and a short cytoplasmic tail without the protein tyrosine phosphatase binding motif I/VXYXXL present in the inhibitory receptors. Sequence analysis demonstrates that three clusters correspond to the inhibitory receptors of 58 kDa, while two clusters encode activating receptors of 50 kDa. TWD other clusters correspond to the inhibitory receptors of 70 kDa and one cluster encodes genes with sequence homology to one of the two p70 clusters but contains the transmembrane and cytoplasmic domains characteristic of activating receptors. The data are consistent with a genomic organization of the KIR genetic region containing at least three KIR genes encoding receptors for each of the gene products of the HLA class I loci. Alternative mRNA splicing could be responsible for generation of activating or inhibitory receptors with the same extracellular domains. Separate genes encoding receptors with opposite function is, however, an equally likely possibility.

A Panel of Artificial APCs Expressing Prevalent HLA Alleles Permits Generation of Cytotoxic T Cells Specific for Both Dominant and Subdominant Viral Epitopes for Adoptive Therapy

Adoptive transfer of virus-specific T cells can treat infections complicating allogeneic hematopoietic cell transplants. However, autologous APCs are often limited in supply. In this study, we describe a panel of artificial APCs (AAPCs) consisting of murine 3T3 cells transduced to express human B7.1, ICAM-1, and LFA-3 that each stably express one of a series of six common HLA class I alleles. In comparative analyses, T cells sensitized with AAPCs expressing a shared HLA allele or autologous APCs loaded with a pool of 15-mer spanning the sequence of CMVpp65 produced similar yields of HLA-restricted CMVpp65-specific T cells; significantly higher yields could be achieved by sensitization with AAPCs transduced to express the CMVpp65 protein. T cells generated were CD8+, IFN-γ+, and exhibited HLA-restricted CMVpp65-specific cytotoxicity. T cells sensitized with either peptide-loaded or transduced AAPCs recognized epitopes presented by each HLA allele known to be immunogenic in humans. Sensitization with AAPCs also permitted expansion of IFN-γ+ cytotoxic effector cells against subdominant epitopes that were either absent or in low frequencies in T cells sensitized with autologous APCs. This replenishable panel of AAPCs can be used for immediate sensitization and expansion of virus-specific T cells of desired HLA restriction for adoptive immunotherapy. It may be of particular value for recipients of transplants from HLA-disparate donors.

Soluble and membrane-bound interleukin (IL)-15 Rα/IL-15 complexes mediate proliferation of high-avidity central memory CD8(+) T cells for adoptive immunotherapy of cancer and infections.

The lack of persistence of infused T cells is a principal limitation of adoptive immunotherapy in man. Interleukin (IL)‐15 can sustain memory T cell expansion when presented in complex with IL‐15Rα (15Rα/15). We developed a novel in‐vitro system for generation of stable 15Rα/15 complexes. Immunologically quantifiable amounts of IL‐15 were obtained when both IL‐15Rα and IL‐15 genes were co‐transduced in NIH 3T3 fibroblast‐based artificial antigen‐presenting cells expressing human leucocyte antigen (HLA) A:0201, β2 microglobulin, CD80, CD58 and CD54 [A2‐artificial antigen presenting cell (AAPC)] and a murine pro‐B cell line (Baf‐3) (A2‐AAPC15Rα/15and Baf‐315Rα/15). Transduction of cells with IL‐15 alone resulted in only transient expression of IL‐15, with minimal amounts of immunologically detectable IL‐15. In comparison, cells transduced with IL‐15Rα alone (A2‐AAPCRα) demonstrated stable expression of IL‐15Rα; however, when loaded with soluble IL‐15 (sIL‐15), these cells sequestered 15Rα/15 intracellularly and also demonstrated minimal amounts of IL‐15. Human T cells stimulated in vitro against a viral antigen (CMVpp65) in the presence of 15Rα/15 generated superior yields of high‐avidity CMVpp65 epitope‐specific T cells [cytomegalovirus‐cytotoxic T lymphocytes (CMV‐CTLs)] responding to ≤ 10− 13 M peptide concentrations, and lysing targets cells at lower effector : target ratios (1 : 10 and 1 : 100), where sIL‐15, sIL‐2 or sIL‐7 CMV‐CTLs demonstrated minimal or no activity. Both soluble and surface presented 15Rα/15, but not sIL‐15, sustained in‐vitro expansion of CD62L+ and CCR7+ central memory phenotype CMV‐CTLs (TCM). 15Rα/15 complexes represent a potent adjuvant for augmenting the efficacy of adoptive immunotherapy. Such cell‐bound or soluble 15Rα/15 complexes could be developed for use in combination immunotherapy approaches.

TCR-mimic bispecific antibodies targeting LMP2A show potent activity against EBV malignancies

EBV infection is associated with a number of malignancies of clinical unmet need, including Hodgkin lymphoma, nasopharyngeal carcinoma, gastric cancer, and posttransplant lymphoproliferative disease (PTLD), all of which express the EBV protein latent membrane protein 2A (LMP2A), an antigen that is difficult to target by conventional antibody approaches. To overcome this, we utilized phage display technology and a structure-guided selection strategy to generate human T cell receptor-like (TCR-like) monoclonal antibodies with exquisite specificity for the LMP2A-derived nonamer peptide, C426LGGLLTMV434 (CLG), as presented on HLA-A*02:01. Our lead construct, clone 38, closely mimics the native binding mode of a TCR, recognizing residues at position P3-P8 of the CLG peptide. To enhance antitumor potency, we constructed dimeric T cell engaging bispecific antibodies (DiBsAb) of clone 38 and an affinity-matured version clone 38-2. Both DiBsAb showed potent antitumor properties in vitro and in immunodeficient mice implanted with EBV transformed B lymphoblastoid cell lines and human T cell effectors. Clone 38 DiBsAb showed a stronger safety profile compared with its affinity-matured variant, with no activity against EBV- tumor cell lines and a panel of normal tissues, and was less cross-reactive against HLA-A*02:01 cells pulsed with a panel of CLG-like peptides predicted from a proteomic analysis. Clone 38 was also shown to recognize the CLG peptide on other HLA-A*02 suballeles, including HLA-A*02:02, HLA-A*02:04, and HLA-A*02:06, allowing for its potential use in additional populations. Clone 38 DiBsAb is a lead candidate to treat EBV malignancies with one of the strongest safety profiles documented for TCR-like mAbs.

Abstract 4978: Adoptively transferred CMV-specific T-cells recognizing dominant and sub-dominant pp65 epitopes demonstrate improvedin vivoinhibition of tumor xenografts in combination with PD-1 inhibition

Adoptive transfer of transplant donor or third party donor derived CMV-specific T cells (CMV-CTL) can effectively treat CMV infections in HSCT recipients. In clinical trials, infusion of partially matched third party CMV-CTLs, has demonstrated high response rates against persistent CMV infection. T-cells (TC) generated in vitro or directly selected in vivo demonstrate a striking preponderance of specificity for 1-2 immunodominant (ID) epitopes presented by specific HLA alleles. ID epitopes elicit higher TC functional activity in vivo, compared to sub-dominant (SD) epitopes. The relative clinical efficacy of TC directed against ID versus SD epitopes in vivo remains undefined. Agents augmenting activity of TC responsive to SD epitopes are unexplored. When these alleles are co-inherited in humans, epitopes of CMVpp65 presented by HLA A*02:01 are ID over HLA A*24:02 presented epitopes. We describe an in vivo model to assess efficacy of CMV-CTLs using colon carcinoma cells (coca)transduced to express CMVpp65, as a surrogate system. HLA A*02:01 + and A*24:02 + human coca cells were transduced to express CMVpp65 and GFP-firefly luciferase (cocapp65). CMV-CTLs responding to either the A*0201 presented ID NLV epitope (A2-NLV) or the A*24:02 presented SD QYD epitope (A24-QYD) were generated from donors co-inheriting HLA A*02:01 and A*24:02 by in vitro stimulation using NIH 3T3 artificial antigen presenting cells, expressing HLA A*02:01 or A*24:02, B7.1, LFA-3, and ICAM1. Tumor cells (10 5 cells) were injected subcutaneously into groups of 5-6 NSG mice on the R flank, and 10 5 cells from a pp65 expressing melanoma cell line (melpp65), lacking expression of HLA A*02:01 or A*24:02 were injected on the L shoulder as control. 2 Groups each received 10 6 of tetramer + A2-NLV or A24-QYD CMV-CTLs i.v per mouse; one of each CMV-CTL treated group also received 2 i.v doses ( 200µg /dose) of anti-PD1 antibody (Nivolumab-BMS) at day 2 and 7 post CTL infusion. Control groups received IL-2, with or without anti-PD1, or HLA mismatched CMV-CTLs. Tumor growth was monitored by bioluminescent imaging. CMV-CTLs responsive to SD A24-QYD epitope induced significant cocapp65 growth suppression compared to controls, but did not eradicate tumors in any animal. Combined treatment of A24-QYD CMV-CTLs with anti-PD-1 Ab induced complete cocapp65 eradication in 2 of 5 mice, with minute residual tumors in 3 mice. Treatment with ID A2-NLV CMV-CTLs induced complete cocapp65 eradication in 2 of 5 mice, and smaller residual tumors compared to SD A24-QYD CTL treatment. Combined treatment with anti-PD-1 and A2-NLV CMV-CTLs led to complete cocapp65 eradication in 3 of 5 mice, with minute tumors in 2 mice. Taken together, these data provide evidence that blocking the PD-1/PD-L1 interaction may significantly augment the antiviral activity of both ID and SD CMV-CTLs. Citation Format: Aisha N. Hasan, Annamalai Selvakumar, Tzu-Yun Kuo, Richard J. O9Reilly. Adoptively transferred CMV-specific T-cells recognizing dominant and sub-dominant pp65 epitopes demonstrate improved in vivo inhibition of tumor xenografts in combination with PD-1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4978. doi:10.1158/1538-7445.AM2017-4978