Supplemental tables and figures

Table e-1:Disorders in which alemtuzumab has been applied off-label

Indication / References
Transplantation
  • graft-versus-host disease after bone marrow transplantation
/ 1
  • renal allograft transplants
/ 2-4
  • Pancreas and kidney-pancreas allograft transplants
/ 5,6
  • Liver allograft transplants
/ 7,8
  • Heart-lung allograft transplants
/ 9,10
  • Intestinal and multivisceral allograft transplants
/ 11,12
Autoimmune diseases
  • Systemic vasculitis
/ 13
  • Wegener’s granulomatosis
/ 14
  • Autoimmune thrombocytopenic purpura
/ 15
  • Autoimmune neutropenia
/ 16,17
  • Inflammatory arthritis
/ 18
  • Diffuse cutaneous scleroderma
/ 19
  • Panuveitis
/ 20

Table e-2: Pharmacological characteristics and potential modes of action of alemtuzumab

References
Target antigen:
  • CD52, a 12-amino-acid glycosylated GPI-bound membrane protein;
/ 21,22
  • Expression of CD52 on a number of cells derived from the lympho-monocytic cell lineage, including T and Bcells, natural killer (NK) cells, dendritic cells and most monocytes and macrophages.
    By contrast, no CD52 expression on neutrophils and precursors cells of the hematopoetic lineage;
/ 23-25
  • Exact biological function of CD52 not fully understood;
  • Possibly Tcell activation upon binding of CD52;
  • Possibly stimulatory co-factor required for regulatory Tcells (Treg)
/ 26
Immunogenicity:
  • Reduction of potential immunogenicity by cloning onto a human IgG1κ antibody backbone.
/ 27
  • Detectable levels of anti-alemtuzumab serum antibodies in 29% of patients after one year of treatment in CAREMSI and II.
/ 28,29
Pharmacokinetics and –dynamics:
  • In MS no robust data available;
  • in hematological patients, non-linear elimination kinetics with considerable interindividual variations dependent on CD52 expression level.
/ 30
Cellular depletion:
  • by complement activation and
  • antibody-dependent cellular cytotoxicity (ADCC)
/ 25,31,32
Depletion kinetics:
  • Complement-mediated cell lysis of leukemic Bcells within 14 hours after addition of alemtuzumab (experimental in vitro data);
/ 33
Repopulation kinetics and effects on the immune system:
  • Monocytes and Bcells: pre-alemtuzumab levels in the peripheral blood reached approximately 3-6 months after treatment;
  • Bcell levels exceeding baseline levels by 124-165%;
/ 25,34-36
  • CD4+ Tcells: lower limits of normal reached after 12 months (median); return to baseline levels after 61 months (median);
  • CD8+ Tcells: lower limits of normal reached after 11 months (median); reach baseline levels reached after 30 months (median);
/ 25,37
  • Specific enrichment of Tregs in the peripheral blood due to repopulation distinctly before CD4+ and CD8+ Tcells.
/ 25,36,38,39
  • Probably only short-term inhibitory effect on the innate immune system, as suggested by studies in a transgenic mouse model expressing human CD52, and observations made in the CAMMS223 extension study37,40.
/ 41
  • Humoral and cellular reponses to vaccinated infectious antigens retained.
/ 42
Potential modes of action #:
  • Initially proposed: leucocytopenia induced anti-inflammatory effects; unlikely given longlasting efficacy beyond cellular repopulation.

  • Promotion of immune tolerance and suppression of the effector T cell repertoire through early repopulation of naive B lymphocytes and Tregs;
/ 43
  • Possible induction of neuroprotective autoimmunity during immune reconstitution ;
/ 44
  • Detectable production of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) detectable in lymphocytes from alemtuzumab-treated patients (experimental in vitro data).
/ 44

# - The proposed modes of action apparently do not translate into a reparative action in secondary progressive MS in which alemtuzumab appears to diminish inflammatory disease activity but fails to affect disease progression. In contrast, in a post-hoc analysis of the CAMMS223 phaseII study clinical improvement of disability was noted even for patients without demonstrable clinical disease activity at baseline and during the study 44.

- This may however, also result in the so-called immune reconstitution autoimmunity, which has been described in the context of stem cell transplantation and highly active anti-retroviral therapy (HAART) in HIV 45, possibly accounting for the pathogenesis of the secondary autoimmune phenomena described. During reconstitution of the immune system there is an imbalance of the repopulating B and T cells; while CD4+ and CD8+ Tcells are still depleted, Tregs begin to accumulate 25. At the same time naïve Bcells repopulate excessively, and appear to be overactivated 46. Additionally, since CD52 depletion may not be complete, it is hypothesized that primarily autoreactive Tcells are reconstituted, i.e. those that have escaped depletion in the thymus, rather than new Tcells being generated from the thymus 47.

Menge et al, Alemtuzumab in MSSupplemental tables1

Table e-3:Baseline patient demographics and clinical characteristics of the different study populations

Open-label studies / CAMMS223 34 / CAREMSI 28 / CARE-MS II 48
Cambridge-cohort 49 / Hirst et al. 50 / IFNb1a-treated patients / Alemtuzumab-treated patients (pooled data) / IFNb1a-treated patients / Alemtuzumab-treated patients / IFNb1a-treated patients / Alemtuzumab-treated patients
N / 58 / 39 / 111 / 222 / 187 / 376 / 202 / 426 + 170 *
Proportion of RRMS / 38 % (n=22) / 100 % / 100 % / 100 % / 100 % / 100 % / 100 % / 100 %
Age (mean, yrs) / NA / 34 / 33 / 32 / 33 / 33 / 36 / 35
Gender (% female) / 67 % / 62 % / 64 % / 64 % / 65 % / 65 % / 65 % / 67 %
Disease duration (mean, yrs) / RRMS: 2.7; SPMS: 11.2 / 2.9 / 1.4 § / 1.3 § / 1.5 § / 1.7 § / 4.1 § / 3.8 §
Baseline EDSS
(median, range) / RRMS: 4.8 + (1.0-7.5);
SPMS: 5.8 +
(3.5-7.0) / mean 4.45
(0-8.5) / 2.0
(0-3.5) / 2.0 (0-3.5) / 2.0 (0-3.5) / 2.0 (0-4.0) / 2.5 (0-6.0) / 2.5 (0-6.5)
ARR / 2.94 / 2.44 / 1.3 / 1.3 / 1.8 # / 1.8 # / 1.5 # / 1.7 #

ARR – annualized relapse rate; NA – not available; RRMS- relapsing-remitting MS; SPMS – secondary progressive MS;

§ - median; time interval since first MS symptoms;

+ - mean EDSS (range);

- annualized relapse rate not unequivocally reported 34;

# - mean number of relapses in the year prior to study initiation;

* - two treatment arms of alemtuzumab 12 mg and 24 mg, respectively

Menge et al, Alemtuzumab in MSSupplemental tables1

Table e-4:Study endpoints of the open label studies

Cambridge-chohort 49 / Hirst et al. 50
Follow-up (months) / RRMS: mean 19 (range 6-74)
SPMS: mean 81 (± 25) / mean 23 (range 3.5-54)
Patients with RRMS treated with Alemtuzumab / 22 / 39
ARR before Alemtuzumab / 2.2 / 2.5
Clinical Endpoints
ARR at end of study / 0.14 / 0.19
Relapse rate reduction by Alemtuzumab / RRMS: 94 % +
SPMS: 97 % + / 92.3 %
Proportion of relapse-free patients / NA / 78.6% at 1 year
Mean change of EDSS at study end compared to baseline EDSS / RRMS: -1.2 (improvement of disability)
SPMS: +0.2 (disability progression) / 0.36
Proportion of patients with sustained disability progression / NA / 17%
Proportion of patients with freedom of clinical disease / NA / NA

NA – not available; ARR – annualized relapse rate;

§ - pooled alemtuzumab data (12 mg and 24 mg arm) presented, numbers in brackets represent alemtuzumab 12mg arm for better comparability to CAREMSI/II (n=112);

* - only patients in the alemtuzumab 12 mg arm were included in efficacy analysis;

# - mean number of relapses in the year prior to study initiation; - annualized relapse rate not unequivocally reported 34;

+ARR compared to pre-treatment disease activity

Menge et al, Alemtuzumab in MSSupplemental tables1

Figure legend e-1 – Potential modes of action

Upper panel: Autoreactive T cells interact with antigen-presenting cells (APC) and B cells within the peripheral lymphoid organs outside the CNS; after activation, these autoreactive lymphocytes are able to transmigrate across the blood–brain barrier (BBB). In the CNS, autoreactive T cells are reactivated resulting in the production of (pro-inflammatory) effector cytokines, attraction of macrophages and microglia, initiation of autoaggressive attacks by CD8+ T cells as well as antibody production by plasma cells and. In concert, these mechanisms lead to demyelination and subsequently to irreversible axonal injury.

Lower panel: Alemtuzumab rapidly depletes CD52-bearing lymphoid cells from the circulation resulting in a long lasting and marked lymphocytopenia. During the early repopulation phase there is an imbalance in favour of naive B cells and regulatory Tcells (Tregs), that may foster and promote immunological tolerance and a state of neuroprotective autoimmunity within the CNS 43,44. Brain-derived and ciliary neurotrophic factors (BDNF and CNTF) secreted from lymphocytes within the CNS may exert additional neuroprotective or neuroreparative effects 44.

Adapted from 51. Lymphoid cells affected by alemtuzumab treatment are depicted in faint coloration.

e-References

Reference List

e1. Hale G, Jacobs P, Wood L et al. CD52 antibodies for prevention of graft-versus-host disease and graft rejection following transplantation of allogeneic peripheral blood stem cells. Bone Marrow Transplant 2000;26:69-76.

e2. Calne R, Moffatt SD, Friend PJ et al. Campath IH allows low-dose cyclosporine monotherapy in 31 cadaveric renal allograft recipients. Transplantation 1999;68:1613-1616.

e3. Weissenbacher A, Boesmueller C, Brandacher G et al. Alemtuzumab in solid organ transplantation and in composite tissue allotransplantation. Immunotherapy 2010;2:783-790.

e4. Hanaway MJ, Woodle ES, Mulgaonkar S et al. Alemtuzumab induction in renal transplantation. N Engl J Med 2011;364:1909-1919.

e5. Kaufman DB, Leventhal JR, Gallon LG, Parker MA. Alemtuzumab induction and prednisone-free maintenance immunotherapy in simultaneous pancreas-kidney transplantation comparison with rabbit antithymocyte globulin induction - long-term results. Am J Transplant 2006;6:331-339.

e6. Gruessner RW, Kandaswamy R, Humar A et al. Calcineurin inhibitor- and steroid-free immunosuppression in pancreas-kidney and solitary pancreas transplantation. Transplantation 2005;79:1184-1189.

e7. Levitsky J, Thudi K, Ison MG et al. Alemtuzumab induction in non-hepatitis C positive liver transplant recipients. Liver Transpl 2011;17:32-37.

e8. Tzakis AG, Tryphonopoulos P, Kato T et al. Preliminary experience with alemtuzumab (Campath-1H) and low-dose tacrolimus immunosuppression in adult liver transplantation. Transplantation 2004;77:1209-1214.

e9. Reams BD, Musselwhite LW, Zaas DW et al. Alemtuzumab in the treatment of refractory acute rejection and bronchiolitis obliterans syndrome after human lung transplantation. Am J Transplant 2007;7:2802-2808.

e10. Das B, Shoemaker L, Recto M et al. Alemtuzumab (Campath-1H) induction in a pediatric heart transplant: successful outcome and rationale for its use. J Heart Lung Transplant 2008;27:242-244.

e11. Tzakis AG, Kato T, Nishida S et al. Alemtuzumab (Campath-1H) combined with tacrolimus in intestinal and multivisceral transplantation. Transplantation 2003;75:1512-1517.

e12. Abu-Elmagd KM, Costa G, Bond GJ et al. Five hundred intestinal and multivisceral transplantations at a single center: major advances with new challenges. Ann Surg 2009;250:567-581.

e13. Mathieson PW, Cobbold SP, Hale G et al. Monoclonal-antibody therapy in systemic vasculitis. N Engl J Med 1990;323:250-254.

e14. Lockwood CM, Thiru S, Stewart S et al. Treatment of refractory Wegener's granulomatosis with humanized monoclonal antibodies. QJM 1996;89:903-912.

e15. Lim SH, Hale G, Marcus RE et al. CAMPATH-1 monoclonal antibody therapy in severe refractory autoimmune thrombocytopenic purpura. Br J Haematol 1993;84:542-544.

e16. Killick SB, Marsh JC, Hale G et al. Sustained remission of severe resistant autoimmune neutropenia with Campath-1H. Br J Haematol 1997;97:306-308.

e17. Gomez-Almaguer D, Solano-Genesta M, Tarin-Arzaga L et al. Low-dose rituximab and alemtuzumab combination therapy for patients with steroid-refractory autoimmune cytopenias. Blood 2010;116:4783-4785.

e18. Watts RA, Isaacs JD, Hale G et al. CAMPATH-1H in inflammatory arthritis. Clin Exp Rheumatol 1993;11 Suppl 8:S165-S167.

e19. Isaacs JD, Hazleman BL, Chakravarty K et al. Monoclonal antibody therapy of diffuse cutaneous scleroderma with CAMPATH-1H. J Rheumatol 1996;23:1103-1106.

e20. Isaacs JD, Hale G, Waldmann H et al. Monoclonal antibody therapy of chronic intraocular inflammation using Campath-1H. Br J Ophthalmol 1995;79:1054-1055.

e21. Xia MQ, Tone M, Packman L et al. Characterization of the CAMPATH-1 (CDw52) antigen: biochemical analysis and cDNA cloning reveal an unusually small peptide backbone. Eur J Immunol 1991;21:1677-1684.

e22. Hale G, Rye PD, Warford A et al. The glycosylphosphatidylinositol-anchored lymphocyte antigen CDw52 is associated with the epididymal maturation of human spermatozoa. J Reprod Immunol 1993;23:189-205.

e23. Ratzinger G, Reagan JL, Heller G et al. Differential CD52 expression by distinct myeloid dendritic cell subsets: implications for alemtuzumab activity at the level of antigen presentation in allogeneic graft-host interactions in transplantation. Blood 2003;101:1422-1429.

e24. Buggins AG, Mufti GJ, Salisbury J et al. Peripheral blood but not tissue dendritic cells express CD52 and are depleted by treatment with alemtuzumab. Blood 2002;100:1715-1720.

e25. Coles AJ, Cox A, le Page E et al. The window of therapeutic opportunity in multiple sclerosis: evidence from monoclonal antibody therapy. J Neurol 2006;253:98-108.

e26. Rowan WC, Hale G, Tite JP, Brett SJ. Cross-linking of the CAMPATH-1 antigen (CD52) triggers activation of normal human T lymphocytes. Int Immunol 1995;7:69-77.

e27. Riechmann L, Clark M, Waldmann H, Winter G. Reshaping human antibodies for therapy. Nature 1988;332:323-327.

e28. Cohen JA, Coles AJ, Arnold DL et al. Alemtuzumab versus interferon beta 1a as first-line treatment for patients with relapsing-remitting multiple sclerosis: a randomised controlled phase 3 trial. Lancet 2012;380:1819-1828.

e29. Keating MJ, Flinn I, Jain V et al. Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study. Blood 2002;99:3554-3561.

e30. Chakraverty R, Orti G, Roughton M et al. Impact of in vivo alemtuzumab dose before reduced intensity conditioning and HLA-identical sibling stem cell transplantation: pharmacokinetics, GVHD, and immune reconstitution. Blood 2010;116:3080-3088.

e31. Hale G, Cobbold SP, Waldmann H et al. Isolation of low-frequency class-switch variants from rat hybrid myelomas. J Immunol Methods 1987;103:59-67.

e32. Hale G, Bright S, Chumbley G et al. Removal of T cells from bone marrow for transplantation: a monoclonal antilymphocyte antibody that fixes human complement. Blood 1983;62:873-882.

e33. Bologna L, Gotti E, Manganini M et al. Mechanism of action of type II, glycoengineered, anti-CD20 monoclonal antibody GA101 in B-chronic lymphocytic leukemia whole blood assays in comparison with rituximab and alemtuzumab. J Immunol 2011;186:3762-3769.

e34. Coles AJ, Compston DA, Selmaj KW et al. Alemtuzumab vs. interferon beta-1a in early multiple sclerosis. N Engl J Med 2008;359:1786-1801.

e35. Hill-Cawthorne GA, Button T, Tuohy O et al. Long term lymphocyte reconstitution after alemtuzumab treatment of multiple sclerosis. J Neurol Neurosurg Psychiatry 2012;83:298-304.

e36. Cossburn MD, Harding K, Ingram G et al. Clinical relevance of differential lymphocyte recovery after alemtuzumab therapy for multiple sclerosis. Neurology 2013;80:55-61.

e37. Coles AJ, Fox E, Vladic A et al. Alemtuzumab more effective than interferon beta-1a at 5-year follow-up of CAMMS223 clinical trial. Neurology 2012;78:1069-1078.

e38. Zhang X, Tao Y, Chopra M et al. Differential Reconstitution of T Cell Subsets following Immunodepleting Treatment with Alemtuzumab (Anti-CD52 Monoclonal Antibody) in Patients with Relapsing-Remitting Multiple Sclerosis. J Immunol 2013.

e39. Havari E, Turner MJ, Campos-Rivera J et al. Impact of alemtuzumab treatment on the survival and function of human regulatory T cells in vitro. Immunology 2013.

e40. Turner M, LaMorte M, Stockman A et al. Analysis of Innate Immune Cells Following Alemtuzumab Treatment in Human CD52 Transgenic Mice. Neurology 2011;76:A140.

e41. Hu Y, Turner MJ, Shields J et al. Investigation of the mechanism of action of alemtuzumab in a human CD52 transgenic mouse model. Immunology 2009;128:260-270.

e42. McCarthy CL, Tuohy O, Compston DA et al. Immune competence after alemtuzumab treatment of multiple sclerosis. Neurology 2013;81:872-876.

e43. Bielekova B, Becker BL. Monoclonal antibodies in MS: mechanisms of action. Neurology 2010;74 Suppl 1:S31-S40.

e44. Jones JL, Anderson JM, Phuah CL et al. Improvement in disability after alemtuzumab treatment of multiple sclerosis is associated with neuroprotective autoimmunity. Brain 2010;133:2232-2247.

e45. Weetman A. Immune reconstitution syndrome and the thyroid. Best Pract Res Clin Endocrinol Metab 2009;23:693-702.

e46. Thompson SA, Jones JL, Cox AL et al. B-cell reconstitution and BAFF after alemtuzumab (Campath-1H) treatment of multiple sclerosis. J Clin Immunol 2010;30:99-105.

e47. Kieseier BC, Wiendl H. The immune repertoire in MS with Alemtuzumab. Nat Rev Neurol 2013;in press.

e48. Coles AJ, Twyman CL, Arnold DL et al. Alemtuzumab for patients with relapsing multiple sclerosis after disease-modifying therapy: a randomised controlled phase 3 trial. Lancet 2012;380:1829-1839.

e49. Coles A, Deans J, Compston A. Campath-1H treatment of multiple sclerosis: lessons from the bedside for the bench. Clin Neurol Neurosurg 2004;106:270-274.

e50. Hirst CL, Pace A, Pickersgill TP et al. Campath 1-H treatment in patients with aggressive relapsing remitting multiple sclerosis. Journal of Neurology 2008;255:231-238.

e51. Menge T and Kieseier BC. Alemtuzumab: Current Concepts and Application for the Treatment of Multiple Sclerosis. CML – Multiple Sclerosis 2011;3:89–104.