After completing his PhD in Pharmacology at the University of Alberta in 1998, Dr. Robert Holt conducted his post-doctoral research at the State University of New York in Albany, NY. He joined the faculty at the University of British Columbia in 2003 where he is now a Professor in the Department of Medical Genetics, a Faculty Member of the Genome Science and Technology program, the Interdisciplinary Oncology Graduate Program, the Genetics Graduate Program and the Health Brain Research Centre. He is also a Professor in the Molecular Biology and Biochemistry department at Simon Fraser University, a Distinguished Scientists at Canada’s Michael Smith Genome Sciences Centre and Co-Director of the BC Cancer Immunotherapy Program.   

With over 200 publications and approximately 70,000 citations, Dr. Holt was listed in the top 1% of Web of Science cited researchers by Clarivate Analytics in 2018. He was the recipient of an Innovation and Achievement Award from BC Biotech in 2004, a Genome BC Award for Scientific Excellence in 2010 and an Excellence in BC Health Care Award in 2014.  

Dr Holt’s work is focused on the structural diversity of T cell and B cell receptors which enable our bodies to discriminate our own cells from a diverse array of pathogens. His group is focused on exploiting our knowledge of T cell and B cell receptors to boost the anti-cancer immune response. This includes engineering T cells to deliver cytotoxic payloads to enhance the immune-mediated killing of cancer cells, and optimizing procedures for isolating, expanding, activating, and then delivering T cells to the body as a targeted immunotherapy for cancer.

Another branch of Dr Holt’s work concerns the relationship between pathogens and cancer; their work in colorectal carcinomas revealed a strong link between colorectal carcinoma and infection with the bacteria Fusobacterium nucleatum, paving the way for targeting this pathogen as a potential preventative measure against colorectal cancer.

Affiliations
  • Co-director, BC Cancer Immunotherapy Program 
  • Professor of Medical Genetics, University of British Columbia 
  • Professor of Molecular Biology & Biochemistry, Simon Fraser University
Credentials
  • B.Sc. Biology, University of British Columbia, 1992
  • Ph.D. Pharmacology, University of Alberta, 1998

Projects

Immunogenetics

the Holt Lab is using deep sequencing methods to explore the role of T cells in cancer, and how to enhance the anti-cancer immune response. They are particularly focused on developing new sequence-based approaches to T cell antigen discovery and characterization.

Cancer Genomes

Dr. Holt’s lab is using deep sequencing and novel computational methods to identify the spectrum of somatic mutations in various cancers, with a particular focus on tumour evolution and the identification of antigens for cancer vaccines.

Synthetic immunology

Cancer immunotherapies using engineered autologous T cells have shown remarkable efficacy against some cancer. Dr. Holt’s team is engineering T cells to selectively deliver modified cytotoxic payloads and pro-drug activators for the purpose of enhanced tumour cell killing and overcoming immune resistance.

Immunotherapy

Selected Publications

The genome sequence of the malaria mosquito Anopheles gambiae.

Science (New York, N.Y.), 2002
Holt, Robert A, Subramanian, G Mani, Halpern, Aaron, Sutton, Granger G, Charlab, Rosane, Nusskern, Deborah R, Wincker, Patrick, Clark, Andrew G, Ribeiro, José M C, Wides, Ron, Salzberg, Steven L, Loftus, Brendan, Yandell, Mark, Majoros, William H, Rusch, Douglas B, Lai, Zhongwu, Kraft, Cheryl L, Abril, Josep F, Anthouard, Veronique, Arensburger, Peter, Atkinson, Peter W, Baden, Holly, de Berardinis, Veronique, Baldwin, Danita, Benes, Vladimir, Biedler, Jim, Blass, Claudia, Bolanos, Randall, Boscus, Didier, Barnstead, Mary, Cai, Shuang, Center, Angela, Chaturverdi, Kabir, Christophides, George K, Chrystal, Mathew A, Clamp, Michele, Cravchik, Anibal, Curwen, Val, Dana, Ali, Delcher, Art, Dew, Ian, Evans, Cheryl A, Flanigan, Michael, Grundschober-Freimoser, Anne, Friedli, Lisa, Gu, Zhiping, Guan, Ping, Guigo, Roderic, Hillenmeyer, Maureen E, Hladun, Susanne L, Hogan, James R, Hong, Young S, Hoover, Jeffrey, Jaillon, Olivier, Ke, Zhaoxi, Kodira, Chinnappa, Kokoza, Elena, Koutsos, Anastasios, Letunic, Ivica, Levitsky, Alex, Liang, Yong, Lin, Jhy-Jhu, Lobo, Neil F, Lopez, John R, Malek, Joel A, McIntosh, Tina C, Meister, Stephan, Miller, Jason, Mobarry, Clark, Mongin, Emmanuel, Murphy, Sean D, O'Brochta, David A, Pfannkoch, Cynthia, Qi, Rong, Regier, Megan A, Remington, Karin, Shao, Hongguang, Sharakhova, Maria V, Sitter, Cynthia D, Shetty, Jyoti, Smith, Thomas J, Strong, Renee, Sun, Jingtao, Thomasova, Dana, Ton, Lucas Q, Topalis, Pantelis, Tu, Zhijian, Unger, Maria F, Walenz, Brian, Wang, Aihui, Wang, Jian, Wang, Mei, Wang, Xuelan, Woodford, Kerry J, Wortman, Jennifer R, Wu, Martin, Yao, Alison, Zdobnov, Evgeny M, Zhang, Hongyu, Zhao, Qi, Zhao, Shaying, Zhu, Shiaoping C, Zhimulev, Igor, Coluzzi, Mario, della Torre, Alessandra, Roth, Charles W, Louis, Christos, Kalush, Francis, Mural, Richard J, Myers, Eugene W, Adams, Mark D, Smith, Hamilton O, Broder, Samuel, Gardner, Malcolm J, Fraser, Claire M, Birney, Ewan, Bork, Peer, Brey, Paul T, Venter, J Craig, Weissenbach, Jean, Kafatos, Fotis C, Collins, Frank H, Hoffman, Stephen L
Anopheles gambiae is the principal vector of malaria, a disease that afflicts more than 500 million people and causes more than 1 million deaths each year. Tenfold shotgun sequence coverage was obtained from the PEST strain of A. gambiae and assembled into scaffolds that span 278 million base pairs. A total of 91% of the genome was organized in 303 scaffolds; the largest scaffold was 23.1 million base pairs. There was substantial genetic variation within this strain, and the apparent existence of two haplotypes of approximately equal frequency ("dual haplotypes") in a substantial fraction of the genome likely reflects the outbred nature of the PEST strain. The sequence produced a conservative inference of more than 400,000 single-nucleotide polymorphisms that showed a markedly bimodal density distribution. Analysis of the genome sequence revealed strong evidence for about 14,000 protein-encoding transcripts. Prominent expansions in specific families of proteins likely involved in cell adhesion and immunity were noted. An expressed sequence tag analysis of genes regulated by blood feeding provided insights into the physiological adaptations of a hematophagous insect.

Profiling the T-cell receptor beta-chain repertoire by massively parallel sequencing.

Genome research, 2009
Freeman, J Douglas, Warren, René L, Webb, John R, Nelson, Brad H, Holt, Robert A
T-cell receptor (TCR) genomic loci undergo somatic V(D)J recombination, plus the addition/subtraction of nontemplated bases at recombination junctions, in order to generate the repertoire of structurally diverse T cells necessary for antigen recognition. TCR beta subunits can be unambiguously identified by their hypervariable CDR3 (Complement Determining Region 3) sequence. This is the site of V(D)J recombination encoding the principal site of antigen contact. The complexity and dynamics of the T-cell repertoire remain unknown because the potential repertoire size has made conventional sequence analysis intractable. Here, we use 5'-RACE, Illumina sequencing, and a novel short read assembly strategy to sample CDR3(beta) diversity in human T lymphocytes from peripheral blood. Assembly of 40.5 million short reads identified 33,664 distinct TCR(beta) clonotypes and provides precise measurements of CDR3(beta) length diversity, usage of nontemplated bases, sequence convergence, and preferences for TRBV (T-cell receptor beta variable gene) and TRBJ (T-cell receptor beta joining gene) gene usage and pairing. CDR3 length between conserved residues of TRBV and TRBJ ranged from 21 to 81 nucleotides (nt). TRBV gene usage ranged from 0.01% for TRBV17 to 24.6% for TRBV20-1. TRBJ gene usage ranged from 1.6% for TRBJ2-6 to 17.2% for TRBJ2-1. We identified 1573 examples of convergence where the same amino acid translation was specified by distinct CDR3(beta) nucleotide sequences. Direct sequence-based immunoprofiling will likely prove to be a useful tool for understanding repertoire dynamics in response to immune challenge, without a priori knowledge of antigen.

Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma.

Genome research, 2012
Castellarin, Mauro, Warren, René L, Freeman, J Douglas, Dreolini, Lisa, Krzywinski, Martin, Strauss, Jaclyn, Barnes, Rebecca, Watson, Peter, Allen-Vercoe, Emma, Moore, Richard A, Holt, Robert A
An estimated 15% or more of the cancer burden worldwide is attributable to known infectious agents. We screened colorectal carcinoma and matched normal tissue specimens using RNA-seq followed by host sequence subtraction and found marked over-representation of Fusobacterium nucleatum sequences in tumors relative to control specimens. F. nucleatum is an invasive anaerobe that has been linked previously to periodontitis and appendicitis, but not to cancer. Fusobacteria are rare constituents of the fecal microbiota, but have been cultured previously from biopsies of inflamed gut mucosa. We obtained a Fusobacterium isolate from a frozen tumor specimen; this showed highest sequence similarity to a known gut mucosa isolate and was confirmed to be invasive. We verified overabundance of Fusobacterium sequences in tumor versus matched normal control tissue by quantitative PCR analysis from a total of 99 subjects (p = 2.5 × 10(-6)), and we observed a positive association with lymph node metastasis.

A survey of genes modulated by host cell infection.

Microbial genomics, 2020
Cochrane, Kyla, Robinson, Avery V, Holt, Robert A, Allen-Vercoe, Emma
Here, we report comprehensive transcriptomic profiles from under conditions that mimic the first stages of bacterial infection in a highly differentiated adenocarcinoma epithelial cell line. Our transcriptomic adenocarcinoma approach allows us to measure the expression dynamics and regulation of bacterial virulence and response factors in real time, and is a novel strategy for clarifying the role of infection in colorectal cancer (CRC) progression. Our data show that: (i) infection alters metabolic and functional pathways in , allowing the bacterium to adapt to the host-imposed milieu; (ii) infection also stimulates the expression of genes required to help induce and promote a hypoxic and inflammatory microenvironment in the host; and (iii) invasion occurs by a haematogenous route of infection. Our study identifies novel gene targets from that are activated during invasion and which may aid in determining how this species invades and promotes disease within the human gastrointestinal tract. These invasion-specific genes may be useful as biomarkers for CRC progression in a host and could also assist in the development of new diagnostic tools and treatments (such as vaccines or small molecule drug targets), which will be able to combat infection and inflammation in the host while circumventing the potential problem of tolerization.

Rapid selection and identification of functional CD8+ T cell epitopes from large peptide-coding libraries.

Nature communications, 2019
Sharma, Govinda, Rive, Craig M, Holt, Robert A
Cytotoxic CD8{{sup}}+{{/sup}} T cells recognize and eliminate infected or malignant cells that present peptide epitopes derived from intracellularly processed antigens on their surface. However, comprehensive profiling of specific major histocompatibility complex (MHC)-bound peptide epitopes that are naturally processed and capable of eliciting a functional T cell response has been challenging. Here, we report a method for deep and unbiased T cell epitope profiling, using in vitro co-culture of CD8{{sup}}+{{/sup}} T cells together with target cells transduced with high-complexity, epitope-encoding minigene libraries. Target cells that are subject to cytotoxic attack from T cells in co-culture are isolated prior to apoptosis by fluorescence-activated cell sorting, and characterized by sequencing the encoded minigenes. We then validate this highly parallelized method using known murine T cell receptor/peptide-MHC pairs and diverse minigene-encoded epitope libraries. Our data thus suggest that this epitope profiling method allows unambiguous and sensitive identification of naturally processed and MHC-presented peptide epitopes.

Risks and Benefits of Chimeric Antigen Receptor T-Cell (CAR-T) Therapy in Cancer: A Systematic Review and Meta-Analysis.

Transfusion medicine reviews, 2019
Grigor, Emma J M, Fergusson, Dean, Kekre, Natasha, Montroy, Joshua, Atkins, Harold, Seftel, Matthew D, Daugaard, Mads, Presseau, Justin, Thavorn, Kednapa, Hutton, Brian, Holt, Robert A, Lalu, Manoj M
Promising efficacy results of chimeric antigen receptor (CAR) T-cell therapy have been tempered by safety considerations. Our objective was to comprehensively summarize the efficacy and safety of CAR-T cell therapy in patients with relapsed or refractory hematologic or solid malignancies. MEDLINE, Embase, and the Cochrane Register of Controlled Trials (inception - November 21, 2017). Interventional studies investigating CAR-T cell therapy in patients with malignancies were included. Our primary outcome of interest was complete response (defined as the absence of detectable cancer). Two independent reviewers extracted relevant data, assessed risk of bias, and graded the quality of evidence using established methods. A total of 42 hematological malignancy studies and 18 solid tumor studies met were included (913 participants). Of 486 evaluable hematologic patients, 54.4% [95% CI, 42.5%-65.9%] experienced complete response in 27 CD19 CAR-T cell therapy studies. Of 65 evaluable hematologic patients, 24.4% [95% CI, 9.4%-50.3%] experienced complete response in seven non-CD19 CAR-T cell therapy studies. Cytokine release syndrome was experienced by 55.3% [95% CI, 40.3%-69.4%] of patients and neurotoxicity 37.2% [95% CI, 28.6%-46.8%] of patients with hematologic malignancies. Of 86 evaluable solid tumor patients, 4.1% [95% CI, 1.6%-10.6%] experienced complete response in eight CAR-T cell therapy studies. Limitations include heterogeneity of study populations, as well as high risk of bias of included studies. There was a strong signal for efficacy of CAR-T cell therapy in patients with CD19+ hematologic malignancies and no overall signal in solid tumor trials published to date. These results will help inform patients, physicians, and other stakeholders of the benefits and risks associated with CAR-T cell therapy.

Twenty-Seven Tamoxifen-Inducible iCre-Driver Mouse Strains for Eye and Brain, Including Seventeen Carrying a New Inducible-First Constitutive-Ready Allele.

Genetics, 2019
Korecki, Andrea J, Hickmott, Jack W, Lam, Siu Ling, Dreolini, Lisa, Mathelier, Anthony, Baker, Oliver, Kuehne, Claudia, Bonaguro, Russell J, Smith, Jillian, Tan, Chin-Vern, Zhou, Michelle, Goldowitz, Daniel, Deussing, Jan M, Stewart, A Francis, Wasserman, Wyeth W, Holt, Robert A, Simpson, Elizabeth M
To understand gene function, the cre/loxP conditional system is the most powerful available for temporal and spatial control of expression in mouse. However, the research community requires more cre recombinase expressing transgenic mouse strains (cre-drivers) that restrict expression to specific cell types. To address these problems, a high-throughput method for large-scale production that produces high-quality results is necessary. Further, endogenous promoters need to be chosen that drive cell type specific expression, or we need to further focus the expression by manipulating the promoter. Here we test the suitability of using knock-ins at the docking site 5' of for rapid development of numerous cre-driver strains focused on expression in adulthood, using an improved cre tamoxifen inducible allele (icre/ERT2), and testing a novel inducible-first, constitutive-ready allele (icre/f3/ERT2/f3). In addition, we test two types of promoters either to capture an endogenous expression pattern (MaxiPromoters), or to restrict expression further using minimal promoter element(s) designed for expression in restricted cell types (MiniPromoters). We provide new cre-driver mouse strains with applicability for brain and eye research. In addition, we demonstrate the feasibility and applicability of using the locus 5' of for the rapid generation of substantial numbers of cre-driver strains. We also provide a new inducible-first constitutive-ready allele to further speed cre-driver generation. Finally, all these strains are available to the research community through The Jackson Laboratory.

Neoantigen characteristics in the context of the complete predicted MHC class I self-immunopeptidome.

Oncoimmunology, 2019
Brown, Scott D, Holt, Robert A
The self-immunopeptidome is the repertoire of all self-peptides that can be presented by the combination of MHC variants carried by an individual, defined by their HLA genotype. Each MHC variant presents a distinct set of self-peptides, and the number of peptides in a set is variable. Subjects carrying MHC variants that present fewer self-peptides should also present fewer mutated peptides, resulting in decreased immune pressure on tumor cells. To explore this, we predicted peptide-MHC binding values using all unique 8-11mer human peptides in the human proteome and all available HLA class I allelic variants, for a total of 134 billion unique peptide--MHC binding predictions. From these predictions, we observe that most peptides are able to be presented by relatively few (< 250) MHC, while some can be presented by upwards of 1,500 different MHC. There is substantial overlap among the repertoires of peptides presented by different MHC and no relationship between the number of peptides presented and HLA population frequency. Nearly 30% of self-peptides are presentable by at least one MHC, leaving 70% of the human peptidome unsurveyed by T cells. We observed similar distributions of predicted self-immunopeptidome sizes in cancer subjects compared to controls, and within the pan-cancer population, predicted self-immunopeptidome size combined with mutational load to predict survival. Self-immunopeptidome analysis revealed evidence for tumor immunoediting and identified specific peptide positions that most influence immunogenicity. Because self-immunopeptidome size is defined by HLA genotypes and approximates neoantigen load, HLA genotyping could offer a rapid predictive biomarker for response to immunotherapy.

A library-based screening method identifies neoantigen-reactive T cells in peripheral blood prior to relapse of ovarian cancer.

Oncoimmunology, 2017
Martin, Spencer D, Wick, Darin A, Nielsen, Julie S, Little, Nicole, Holt, Robert A, Nelson, Brad H
Mutated cancer antigens, or neoantigens, represent compelling immunological targets and appear to underlie the success of several forms of immunotherapy. While there are anecdotal reports of neoantigen-specific T cells being present in the peripheral blood and/or tumors of cancer patients, effective adoptive cell therapy (ACT) against neoantigens will require reliable methods to isolate and expand rare, neoantigen-specific T cells from clinically available biospecimens, ideally prior to clinical relapse. Here, we addressed this need using "mini-lines", large libraries of parallel T cell cultures, each originating from only 2,000 T cells. Using small quantities of peripheral blood from multiple time points in an ovarian cancer patient, we screened over 3.3 × 10{{sup}}6{{/sup}} CD8{{sup}}+{{/sup}} T cells by ELISPOT for recognition of peptides corresponding to the full complement of somatic mutations (n = 37) from the patient's tumor. We identified ten T cell lines which collectively recognized peptides encoding five distinct mutations. Six of the ten T cell lines recognized a previously described neoantigen from this patient (HSDL1{{sup}}L25V{{/sup}}), whereas the remaining four lines recognized peptides corresponding to four other mutations. Only the HSDL1{{sup}}L25V{{/sup}}-specific T cell lines recognized autologous tumor. HSDL1{{sup}}L25V{{/sup}}-specific T cells comprised at least three distinct clonotypes and could be identified and expanded from peripheral blood 3-9 months prior to the first tumor recurrence. These T cells became undetectable at later time points, underscoring the dynamic nature of the response. Thus, neoantigen-specific T cells can be expanded from small volumes of blood during tumor remission, making pre-emptive ACT a plausible clinical strategy.
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