Antigen Receptors Prevent Antigen Escape by Malignant B Cells

The adoptive transfer of T cells expressing anti-CD19 chimeric antigen receptors (CARs) has shown remarkable curative potential against advanced B-cell malignancies, butmultiple trials have also reported patient relapses due to the emergence of CD19-negative leukemic cells. Here, we report the design and optimization of single-chain, bispecificCARs that trigger robust cytotoxicity against target cells expressing either CD19 or CD20, two clinically validated targets for B-cell malignancies.We determined the structural parameters required for efficient dual-antigen recognition, and we demonstrate thatoptimizedbispecificCARs can controlbothwildtype B-cell lymphoma and CD19 mutants with equal efficiency in vivo. To our knowledge, this is the first bispecific CAR capable of preventing antigen escape by performing true OR-gate signal computation on a clinically relevant pair of tumor-associated antigens. The CD19-OR-CD20 CAR is fully compatible with existing T-cell manufacturing procedures and implementable by current clinical protocols. These results present an effective solution to the challenge of antigen escape in CD19 CAR T-cell therapy, and they highlight the utility of structure-based rational design in thedevelopmentof receptorswithhigher-level complexity. Cancer Immunol Res; 4(6); 498–508. 2016 AACR. See related Spotlight by Sadelain, p. 473.

[1]  Y. Chen,et al.  T Cells Expressing CD19/CD20 Bispecific Chimeric Antigen Receptors Prevent Antigen Escape by Malignant B Cells , 2016, Cancer Immunology Research.

[2]  J. E. Brewer,et al.  NY-ESO-1 specific TCR engineered T-cells mediate sustained antigen-specific antitumor effects in myeloma , 2015, Nature Medicine.

[3]  R. Krance,et al.  Inducible caspase-9 suicide gene controls adverse effects from alloreplete T cells after haploidentical stem cell transplantation. , 2015, Blood.

[4]  P. Silver,et al.  Identification and selective expansion of functionally superior T cells expressing chimeric antigen receptors , 2015, Journal of Translational Medicine.

[5]  S. Rosenberg,et al.  Adoptive cell transfer as personalized immunotherapy for human cancer , 2015, Science.

[6]  R. Kaplan,et al.  4-1BB Costimulation Ameliorates T Cell Exhaustion Induced by Tonic Signaling of Chimeric Antigen Receptors , 2015, Nature Medicine.

[7]  Sadik H. Kassim,et al.  Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. , 2015, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[8]  Seth M Steinberg,et al.  T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial , 2015, The Lancet.

[9]  Matthew J. Frigault,et al.  Identification of Chimeric Antigen Receptors That Mediate Constitutive or Inducible Proliferation of T Cells , 2015, Cancer Immunology Research.

[10]  K. Kelly-Spratt,et al.  Functional Tuning of CARs Reveals Signaling Threshold above Which CD8+ CTL Antitumor Potency Is Attenuated due to Cell Fas–FasL-Dependent AICD , 2015, Cancer Immunology Research.

[11]  C. June,et al.  Going viral: chimeric antigen receptor T‐cell therapy for hematological malignancies , 2015, Immunological reviews.

[12]  Pamela A Shaw,et al.  Chimeric antigen receptor T cells for sustained remissions in leukemia. , 2014, The New England journal of medicine.

[13]  S. Riddell,et al.  The Nonsignaling Extracellular Spacer Domain of Chimeric Antigen Receptors Is Decisive for In Vivo Antitumor Activity , 2014, Cancer Immunology Research.

[14]  Pradip Bajgain,et al.  Kinetics of tumor destruction by chimeric antigen receptor-modified T cells. , 2014, Molecular therapy : the journal of the American Society of Gene Therapy.

[15]  Qing He,et al.  Efficacy and Toxicity Management of 19-28z CAR T Cell Therapy in B Cell Acute Lymphoblastic Leukemia , 2014, Science Translational Medicine.

[16]  T. Weimer,et al.  Pharmacological characteristics of a novel, recombinant fusion protein linking coagulation factor VIIa with albumin (rVIIa-FP) , 2014, Journal of thrombosis and haemostasis : JTH.

[17]  Michel Sadelain,et al.  PD-1– and CTLA-4–Based Inhibitory Chimeric Antigen Receptors (iCARs) Divert Off-Target Immunotherapy Responses , 2013, Science Translational Medicine.

[18]  Y. Kew,et al.  Combinational Targeting Offsets Antigen Escape and Enhances Effector Functions of Adoptively Transferred T Cells in Glioblastoma , 2013, Molecular therapy : the journal of the American Society of Gene Therapy.

[19]  M. Kalos,et al.  Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology. , 2013, Immunity.

[20]  Matthew L Baker,et al.  TanCAR: A Novel Bispecific Chimeric Antigen Receptor for Cancer Immunotherapy , 2013, Molecular therapy. Nucleic acids.

[21]  Bernd Hauck,et al.  Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. , 2013, The New England journal of medicine.

[22]  Qing He,et al.  CD19-Targeted T Cells Rapidly Induce Molecular Remissions in Adults with Chemotherapy-Refractory Acute Lymphoblastic Leukemia , 2013, Science Translational Medicine.

[23]  Jinjuan Wang,et al.  CD20-specific adoptive immunotherapy for lymphoma using a chimeric antigen receptor with both CD28 and 4-1BB domains: pilot clinical trial results. , 2012, Blood.

[24]  W. Wilson,et al.  B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. , 2012, Blood.

[25]  David L. Porter,et al.  T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish Memory in Patients with Advanced Leukemia , 2011, Science Translational Medicine.

[26]  S. Riddell,et al.  A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. , 2011, Blood.

[27]  C. Heirman,et al.  Large double copy vectors are functional but show a size-dependent decline in transduction efficiency. , 2010, Journal of biotechnology.

[28]  D. G. Gibson,et al.  Enzymatic assembly of DNA molecules up to several hundred kilobases , 2009, Nature Methods.

[29]  Jinjuan Wang,et al.  Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell lymphoma using genetically modified autologous CD20-specific T cells. , 2008, Blood.

[30]  T. Weimer,et al.  Prolonged in-vivo half-life of factor VIIa by fusion to albumin , 2008, Thrombosis and Haemostasis.

[31]  P. Stern,et al.  The Role of Extracellular Spacer Regions in the Optimal Design of Chimeric Immune Receptors: Evaluation of Four Different scFvs and Antigens , 2005, Journal of immunotherapy.

[32]  W. Wriggers,et al.  Conformations of variably linked chimeric proteins evaluated by synchrotron X‐ray small‐angle scattering , 2004, Proteins.

[33]  A. Engert,et al.  An overview of the current clinical use of the anti-CD20 monoclonal antibody rituximab. , 2003, Annals of oncology : official journal of the European Society for Medical Oncology.

[34]  R. Rickert,et al.  CD19 Function in Early and Late B Cell Development: I. Maintenance of Follicular and Marginal Zone B Cells Requires CD19-Dependent Survival Signals1 , 2003, The Journal of Immunology.

[35]  M. Kumar,et al.  Systematic determination of the packaging limit of lentiviral vectors. , 2001, Human gene therapy.

[36]  Teruyuki Nagamune,et al.  Design of the linkers which effectively separate domains of a bifunctional fusion protein. , 2001, Protein engineering.

[37]  S. Steinberg,et al.  Immune selection after antigen-specific immunotherapy of melanoma. , 1999, Surgery.

[38]  A. Wu,et al.  CD20 is a molecular target for scFvFc:zeta receptor redirected T cells: implications for cellular immunotherapy of CD20+ malignancy. , 1998, Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation.

[39]  A. Scott,et al.  Construction and characterisation of a functional CD19 specific single chain Fv fragment for immunotherapy of B lineage leukaemia and lymphoma. , 1997, Molecular immunology.

[40]  F. Oesch,et al.  Inverse relationship of melanocyte differentiation antigen expression in melanoma tissues and CD8+ cytotoxic‐T‐cell responses: Evidence for immunoselection of antigen‐loss variants in vivo , 1996, International journal of cancer.

[41]  T. Tedder,et al.  CD20: a regulator of cell-cycle progression of B lymphocytes. , 1994, Immunology today.

[42]  S. Riddell,et al.  The use of anti-CD3 and anti-CD28 monoclonal antibodies to clone and expand human antigen-specific T cells. , 1990, Journal of immunological methods.

[43]  T. Tedder,et al.  Isolation of cDNAs encoding the CD19 antigen of human and mouse B lymphocytes. A new member of the immunoglobulin superfamily. , 1989, Journal of immunology.

[44]  J. Brown,et al.  Molecular cloning of the human B cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains. , 1988, The EMBO journal.

[45]  G. Lenoir,et al.  Human blymphocytes immortalization by epstein-barr virus in the presence of cyclosporin a , 1986, In Vitro Cellular & Developmental Biology.