Methods for enhancing immunosuppressive therapy by multiple administration of αβTCR-binding polypeptide

#Solid organ transplantation and stem cell transplantation are lifesaving techniques now used on a daily basis around the world. Despite decades of research into immunoprophylaxis, the use of immunosuppressive agents to prevent rejection of these transplants is still far from optimal. Toxicity, opportunistic infections, cytokine storm and an increased risk of cancer are prevalent in patients treated with these agents, who often require lifelong immunosuppression. The use of biologics in this arena has improved patient outcome to some degree, yet these side effects continue to present great difficulties for patients and doctors.

#Alpha/beta T lymphocytes recognize peptide-MHC ligands by means of a multimeric protein ensemble termed the αβ T cell antigen receptor (TCR)-CD3 complex. This structure is composed of a variable αβ TCR dimer which binds antigens, and three invariant dimers (CD3γε, δε, and ζζ) which are involved in TCR-CD3 surface transport, stabilization and signal transduction. The alpha beta T cell receptor (αβ TCR) is expressed on the majority (approximately 95%) of T cells and has a critical role in T cell activation via engagement of antigen displayed on MHC. New humanized monoclonal antibodies targeting the αβTCR cells have been developed that show improved binding profiles (WO2013037484). These promising new antibodies (described both in WO2013037484 and herein) include GL1BM VH31, GL1BM VH28, HEBE1 H66, and HEBE1 H71. There is a need in the art for improved methods of administration of this type of humanized anti-αβTCR antibody which can counter alloreactive T lymphocytes while sparing pathogen inactive T cells (such as γδ T-cells, iNK T cells, and CD3-CD56+ NK cells).

#The present invention is based in part on the surprising discovery that repetitive administration of humanized anti-αβTCR-binding polypeptide to a subject resulted in a significant reduction of αβ T cells relative to γδ T cells in the subject. Importantly, by increasing γδ T cell levels relative to αβ T cells in the subject, one or more advantageous innate and adaptive immune features provided by γδ T cells could be enhanced in the subject. Retaining γδ T cell-mediated immunity is particularly important, e.g., when treating a T-cell mediated disorder in a subject, optionally when using transplanted cells, tissues, organs or the like. The present invention also demonstrates the effectiveness of humanized monoclonal antibodies targeting the αβTCR both in vitro and in vivo. Improved methods for the use monoclonal antibodies targeting the αβTCR are also provided.

#The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

#FIGS. 1A-1B graphically depict the results of flow cytometry studies demonstrating humanized αβ T-cell antibody-induced apoptosis of unstimulated αβ T cells. FIG. 1A graphically depicts pre-apoptotic Annexin V positive αβ T cells given as a percentage of human αβ T cells. FIG. 1B graphically depicts apoptotic Annexin V and 7-AAD double positive PBMC as a percentage of all events.

#FIGS. 2A-2D graphically depict the results of flow cytometry studies demonstrating humanized αβ T-cell antibody-induced apoptosis of activated αβ T cells. FIG. 2A graphically depicts representative Annexin V and 7-AAD staining, for all time points and regimen. FIG. 2B graphically depicts the statistical evaluation of the percentage of Annexin V-positive αβ T cells after different treatments. Error bars present SEM of three independent experiments, *=p<0.05, **=p<0.01. FIG. 2C graphically depicts one of three representative experiments demonstrating the increase of γδ T cells among donor CD3+ T cells in PBMC during incubation with GZ-αβTCR. FIG. 2D graphically depicts cytokine production of IFN-γ, TNF-α, and IL-4 by αβ T cells before and after αβ-mAb treatment.

#The present disclosure provides improved methods for treating T cell-mediated disorders (e.g., graft-versus-host-disease and the like) in which an underlying pathology involves detrimental activation of the subject's immune system. The methods provided herein generally involve administering to a subject in need thereof an effective amount of a humanized binding polypeptide that is specific to the alpha beta T-cell receptor (αβ-TCR).

#Unless otherwise stated, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can be used in the methods of techniques of the present disclosure. All publications cited herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing the methodologies, reagents, and tools reported in the publications that might be used in connection with the disclosure.

#The methods and techniques of the present application are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Gennaro, A. R., ed. (1990) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Hardman, J. G., Limbird, L. E., and Gilman, A. G., eds. (2001) The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill Co.; Colowick, S. et al., eds., Methods In Enzymology, Academic Press, Inc.; Weir, D. M. and Blackwell, C. C., eds. (1986) Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications; Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al., eds. (1999) Short Protocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press; Newton, C. R. and Graham, A., eds. (1997) PCR (Introduction to Biotechniques Series), 2nd ed., Springer-Verlag.