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Cytotoxic Potential In M Ab Therapy

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Impact of Studies Predating Rituximab

Impact of Studies Predating Rituximab

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  • 1. Achieving better cytotoxic potential of NK cells for immune potency & mAb therapy such as Rituxan. Colin M Perrott Engineering of newer anti-CD20 mAbs attempts to increase their ADCC effectiveness substantially, often at the cost of CDC efficiency. Serum can augment NK cell cytotoxicity beyond that of media. We propose this cytotoxic stimulation arises from serum DHEAS via PKC-βII activation and relies on continued enzymatic transformation of cholesterol. 1
  • 2. Abstract We review the broad functions by which Rituxan eliminates B-cells and note that newer mAb’s are engineered to increase the binding affinity required for cell lysis by ADCC, loss in CDC performance notwithstanding. A simple process model that we described recently underscores this logic. It demonstrates also that the final process efficiency achieved via the mAb is necessarily a multiple of the baseline cytotoxity of critical effector cells such NK cells. It is important therefore to understand the spectrum of natural factors that stimulate and also inhibit NK cell cytotoxicity as the processes of aging and also illness ensue. Recently we concluded that enhanced Rituxan induced B-cell lysis in serum likely arises from heightened NK cell cytotoxicity compared to that achieved in vitro with standard laboratory media. We surmised that the androgenic steroid ester DHEAS could be the key stimulant factor. Here, we describe some available research data which shows DHEAS is a powerful stimulator of NK cell cytotoxicity and acts in conjunction with known agents such as IL-2. Furthermore, this effect is opposite the inhibitory role of glucocorticoids. Additionally, DHEAS is known to act beneficially on the NF-kB cellular pathway but via a different mechanism from that of glucocorticoid binding. Given that DHEAS is the most abundant steroid in human serum and declines sharply with advancing age, it is important to determine options for managing this hormone beneficially in the normal course of healthy living - and also as a precursor to mAb therapy such as with Rituxan. Since there are known antagonists to its function, these should be explored in an attempt to achieve a comprehensive model of the impost of age on immune potency. April 2009 Cytotoxic potential in mAb therapy : 2 Colin M Perrott
  • 3. IgG1 is not a stick insect ! Despite the Pictures we see at times. Domains are closely paired – except for CH2 domains near the hinge region. Antigen Binding HINGE Complement Activation Effector Cell Binding Sources: Center for BioMolecular Modeling;http://www.rpc.msoe.edu/cbm2/IgG.html Sharkey & Goldenberg CA Cancer J Clin (2006) April 2009 Cytotoxic potential in mAb therapy : 3 Colin M Perrott
  • 4. INDICATIONS AND USAGE Rituxan® (rituximab) is indicated for single agent treatment of patients with: • Relapsed or refractory, low-grade or follicular, CD20-positive, B-cell NHL • Non-progressing (including stable disease), low-grade, CD20-positive B-cell NHL after first-line CVP http://www.rituxan.com/lymphoma/hcp/MOA/index.m ADCC (antibody-dependent cell-mediated cytotoxicity): Natural killer cells, T cells, and macrophages recognize and kill antibody-labeled target cells, leading to cell lysis April 2009 Cytotoxic potential in mAb therapy : 4 Colin M Perrott
  • 5. INDICATIONS AND USAGE Rituxan® (rituximab) is indicated for combination treatments of: • Previously untreated follicular, CD20-positive, B-cell NHL in combination with CVP chemotherapy • Previously untreated diffuse large B-cell, CD20-positive NHL in combination with CHOP or other anthracycline-based chemotherapy regimens http://www.rituxan.com/lymphoma/hcp/MOA/index.m CDC (complement-dependent cytotoxicity): Binding of the antibody recruits complement proteins, which punch holes in the cell membrane, flooding the cell and leading to cell lysis Apoptosis: Binding of the antibody signals the cell to self-destruct resulting in cell lysis. Apoptosis is regarded as relatively ineffective in actual therapy. April 2009 Cytotoxic potential in mAb therapy : 5 Colin M Perrott
  • 6. CDC and ADCC One process acts on CH2 and the other on CH3 Are they mutually exclusive – given that domains CH2 are not closely paired ? Two regions of the CH2 domain are critical for FcγR’s and complement C1q binding. Mutations made in the CH2 domain can increase both ADCC and CDC Mutations at the interface CH2 / CH3 can increase the half-life of IgG1 Residue material bound into the hinge region has significant effect on performance Simplistic signal analysis: ∇ The CH3 and variable domains communicate via the molecular backbone near the hinge ∇ The detailed electrochemical processes reflect the entire molecular structure of the region ∇ Changes occurring or created at CH2 may influence CH3 signaling and vice-versa Source: Invivogen: http://www.invivogen.com/family.php?ID=164 April 2009 Cytotoxic potential in mAb therapy : 6 Colin M Perrott
  • 7. New anti CD20 monoclonal antibodies for B-cell non-Hodgkin lymphoma GA-101 Glycol-engineered Fc portion and a modified elbow hinge. 50-fold higher binding affinity to the FcRIIIa resulting in a 10 to 100-fold increase in ADCC in vitro. Apoptosis inducer. Reduced CDC effectiveness. AME-133v Binds with an increased affinity to FcRIIIa (CD16): 10-fold increase found in cytotoxicity relative to rituximab in vitro. Phase 1/2 study ongoing for patients with relapsed/refractory follicular lymphoma rhuMAb v114 30-fold greater binding to the low-affinity variant of FcRIII than to rituximab: 2 to 10 times improved ADCC relative to rituximab in vitro. Ofatumumab (HuMaxCD20) Humanized antibody that binds to a different CD20 epitope than rituximab: Phase 1/2 trials demonstrated activity in patients with follicular lymphoma and CLL. IMMU-106 (hA20) Humanized antibody: Phase 1/2 studies showed a 53% ORR in patients with recurrent B- cell NHL, including 6 patients that achieved CR. Source; Bello and Sotomayor (2007) Hematology, 233-242 ADCC, antibody-dependent cell-mediated cytotoxicity; CLL, chronic lymphocytic leukemia; ORR, overall response rate; CR, complete response April 2009 Cytotoxic potential in mAb therapy : 7 Colin M Perrott
  • 8. There are new mAbs in the pipeline. Notice : There is an interaction between expected ADCC and CDC performances. Designs attempt huge proportional changes to the binding affinity FcγR. Why ? The philosophy seeks uniform effectiveness across a broad range of CD20 expression levels from CLL upward. -- But -- Success relies on plentiful stimulated effector cells in vivo. April 2009 Cytotoxic potential in mAb therapy : 8 Colin M Perrott
  • 9. Rituxan lysis of B-cells is parameter sensitive Constituents of serum are very important to the total outcome of therapy MODEL OF RITUXAN LYSIS 1 0.9 0.8 Cell Lysis Probability 0.7 0.6 0.5 0.4 0.3 ADCC in media 0.2 CDC by serum Lysis in Serum 0.1 Idealized Process 0 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 ABS CD20 expression (millions) WM B-cell CD20 expression is in the range ~ 0.3 to 0.6 million/cell April 2009 Cytotoxic potential in mAb therapy : 9 Colin M Perrott
  • 10. There is quite a lot we can understand just from the math The results show that CDC and ADCC do not exclude each other. They are alternatives for cell lysis 10
  • 11. Predicted gain from improved NK cell Cytotoxicity A significant gain occurs from media to serum, but ♣ very big steps are still required from there ! MODEL: CDC + variable ADCC Cytotoxicity 1 0.9 Cell Lysis Probability 0.8 ADCC in media Lysis in Serum 2x Cytotoxicity 0.7 4x Cytotoxicity 16x Cytotoxicity 0.6 0.5 0.4 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 ABS CD20 expression (millions) WM B-cell CD20 expression is in the range ~ 0.3 to 0.6 million/cell April 2009 Cytotoxic potential in mAb therapy : 11 Colin M Perrott
  • 12. Near enough does not fill the gap! Survivor cells may range up to 30% in human serum. They will attempt to proliferate - cell populations always attempt to exploit available resources - the population declines if resources cannot be used - higher initial cell populations show more dramatic response OPPORTUNISTIC GROWTH 1 Bi-level Systems 1 0.8 POPULATIONNêK POPULATION NêK 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0 20 40 60 80 100 PERIOD T 60 80 100 120 140 160 PERIOD T April 2009 Cytotoxic potential in mAb therapy : 12 Colin M Perrott
  • 13. Predicted gain from improved NK cell Cytotoxicity What matters is the number of cells surviving…. ….and the NK cell cytotoxicity in patients’ serum MODEL: CDC + variable ADCC Cytotoxicity 0.6 Cell Survival Probability 0.5 ADCC in media 0.4 Lysis in Serum 2x Cytotoxicity 0.3 4x Cytotoxicity 16x Cytotoxicity 0.2 0.1 0 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 ABS CD20 expression (millions) WM B-cell CD20 expression is in the range ~ 0.3 to 0.6 million/cell April 2009 Cytotoxic potential in mAb therapy : 13 Colin M Perrott
  • 14. What might the cytotoxic enhancement involve? Proposition from in vitro studies: ADCC and CDC operate in a complementary fashion. We concluded: Serum augments the cytotoxity of effector cells ( stimulated NK cells) substantially in comparison to the level achieved by IL-2 based media. Model Features: The enhancement has constant magnitude for all CD20 expression. There is a simple up-scaling of the cytotoxic efficiency of the effector cells as a result of the introduction of human serum. Model Outcomes: Complement is essential to cell lysis at all CD20 expression levels CDC is the prime mechanism at CD20 higher than ~ 650K/cell ADCC provides the baseline performance and is environmentally sensitive Critical Point: Process enhancement by serum is probably due to NK cell cytotoxicity being heightened by a component of the serum… most likely DHEAS April 2009 Cytotoxic potential in mAb therapy : 14 Colin M Perrott
  • 15. The active factor must be plentiful in serum of healthy people It must stimulate NK cells Be an agonistic with IL-2 A logical contender is serum DHEAS Source: Kiechl, S. et al. Arterioscler Thromb Vasc Biol 2000;20:1094-1100 April 2009 Cytotoxic potential in mAb therapy : 15 Colin M Perrott
  • 16. THE CHOLESTEROL ENGINE CHOLESTEROL TNF, IL-1β ACTH IL-1β, TGF-β SULFOTRANSFERASE, Sult2A1 TNF DHEA DHEAS IL-6 DEHYDROEPIANDOSTERONE DHEA SULFATE IL-6 IL-6 SULFATASE TNF PROMOTER SUPPRESSOR ALDOSTERONE CORTISOL THE ENGINE RESPONDS TO HORMONES, AGE & CYTOKINES DHEAS IS AN ABUNDANT PRO-HORMONE IN THE SERUM OF HUMANS AND PRIMATES. RODENT MODELS CAN BE MISLEADING. ACTH = Corticotrophin, a hormone secreted by the anterior pituitary gland All steps in the reaction pathways are dependent on specific enzyme activity. April 2009 Cytotoxic potential in mAb therapy : 16 Colin M Perrott
  • 17. AN OVERVIEW OF LITERATURE; Komaki et al recorded serum DHEAS increase during fasting by non-obese individuals from ~5.5 to ~8.0 μmol/L. This had associated x1.75 increase in spontaneous natural killer (NK) cell cytotoxicity without change in the dominant cytokines (IL-1β, IL-2, IL-6, TNF-α, IFN-γ), in GM-CSF, sIL-2R or corticotrophin. The cytotoxic function (NKCC) of NK cells is regulated by: An autocrine mechanism related to cytokines [e.g. IL-2, IFN-γ, and IFN-β], or Protein kinase C (PKC)-dependent mobilization of proteolytic granules. Solerte et al report dose dependent enhancement of NKCC by DHEAS that correlates positively with locally generated insulin-like growth factor IGF-1. This and other data favor mechanistic action by PKC (specifically PKC-βII isoform) activation. This agrees with other studies of DHEAS effect on the NF-κB pathway. The IGF-1 might also regulate pro-inflammatory cytokine responses contrary to the immunosuppressive action of glucocorticoids. Specificity to the PKC-βII isoform is important. It determines functional outcomes and requires precision in attempts at mediation or interpretation of dynamic effects. In other cell types, PKC-βII and VEGF expression increase concurrently. April 2009 Cytotoxic potential in mAb therapy : 17 Colin M Perrott
  • 18. DHEAS and IL-2 stimulate NK cell cytotoxicity S. B. Solerte et al, Dehydroepiandrosterone Sulfate Enhances Natural Killer Cell Cytotoxicity in Humans Via Locally Generated Immunoreactive Insulin-Like Growth Factor I, J Clin Endocrinol Metab 84: 3260–3267, 1999 Variations in NKCC, expressed as the percent increase in cytotoxicity, after incubation with DHEAS (from 10-8-10-5 mol/L·mL); a) DHEAS plus SST (10-6 mol/L·Ml); b) DHEAS plus IL-2 (100 IU/mL), and; c) IL-2 plus SST. Data (mean ± SD) are related to healthy young (open bars) and healthy elderly (closed bars) subjects. Asterisks denote statistical significance between groups * P<0.05 and ** P<0.01 Patient age had limited effect on NK cell responses. NK cell action generates Immunoreactive Insulin-Like Growth Factor IGF-I. This was suppressed by somatostatin-14 (SST). SST inhibits intracellular signaling via cAMP, acts against growth hormone and many other hormones. It comes from hypothalamic and CNS neurons, pancreatic cells and cells in the GI epithelium. April 2009 Cytotoxic potential in mAb therapy : 18 Colin M Perrott
  • 19. DHEAS and IL-2 stimulate NK cell cytotoxicity Variations in NKCC, expressed as lytic response (LU) after incubation with DHEAS (from 10-8-10-5 mol/L·mL; a), DHEAS plus SST (10-6 mol/L·mL; b), and DHEAS plus IL-2 (100 IU/mL; c). Data (mean ± SD) are related to healthy young (open circles) and healthy elderly (closed circles) subjects. Asterisks denote statistical significance between groups at P < 0.05 (*) and P < 0.01 (**). Solerte, S. B. et al. J Clin Endocrinol Metab 1999;84:3260-3267 NK cells from older patients showed about half the NKCC responsiveness of those from younger patients: x1.50 increase in NKCC for the young and x1.25 increase for the older at the first dosage level for DHEAS. Our Rituxan lysis model required ADCC efficiency increase by x1.43 from 47.2 ±13.8% to 67.6 ± 8.5%. SST suppressed the benefits of both DHEAS and IL-6. April 2009 Cytotoxic potential in mAb therapy : 19 Colin M Perrott
  • 20. Conclusions Our model is consistent with current objectives in mAb development. It underscores the importance of spontaneous cellular cytotoxicity. The data is consistent with NK cell cytotoxicity stimulation by DHEAS in the absence of cytokine or other chemical / hormonal assistance. Age-related or illness induced decline of DHEAS level potentially sets the baseline for ☻ innate immune resilience, and ☻ response to Rituxan or like mAb therapy. SUPPLEMENTARY INFORMATION FOLLOWS 20
  • 21. Complement lysis mechanisms CDC & CDCC CDCC CDC After Catron et al, (2004) doi:10.1182/blood-2004-03-1110 April 2009 Cytotoxic potential in mAb therapy : 21 Colin M Perrott
  • 22. Possible interactions of complement, serum and effector cells ♣ ADCC, ♣ CDCC and ♠ S-ADCC (with DHEAS assist) ADCC S-ADCC ? DHEAS CDCC Modified From Zhou, X. et al. Oncologist 2008;13:954-966 April 2009 Cytotoxic potential in mAb therapy : 22 Colin M Perrott
  • 23. A ROLE FOR PENDANT SPECIES ON THE IgG ? Complement activation occurs at CH2, close to the hinge region. This region is between the reactive ends, CH3 in Fc and CDR’s on Fab. Electrochemical changes might influence flexibility and receptor affinity. Fa Fa b b Fa b b Fa Fc Fc Fab ab F Fc April 2009 Cytotoxic potential in mAb therapy : 23 Colin M Perrott
  • 24. April 2009 Cytotoxic potential in mAb therapy : 24 Colin M Perrott
  • 25. THE CHOLERSTEROL ENGINE IN SOME MORE DETAIL April 2009 Cytotoxic potential in mAb therapy : 25 Colin M Perrott
  • 26. AN INTERESTING STUDY; DHEAS CORRELATES WITH NK-CELL CYTOTOXICITY IN SERA FROM PATIENTS DURING FASTING ( BMI~20 ). NO CHANGES WERE DETECTED IN IL-2 OR OTHER CYTOKINES. G Komaki et al, American Journal of Clinical Nutrition (1997) 66: 147-152 Alterations in lymphocyte subsets and pituitary-adrenal gland-related hormones during fasting We investigated changes in the immunoendocrine system during fasting. Ten hospitalized patients aged 14-46 y with psychosomatic disorders fasted for 7 or 10 d. Blood samples were collected before and on days 3 and 7 of the 7-d fasts. When fasting continued to 10 d, an additional sample was taken on day 10. We measured blood cellularity (white blood cells and total lymphocytes), the total number and percentage of lymphocyte subsets (CD2, CD3, CD4, CD8, and CD19), natural killer (NK) cell activity, cytokines (interleukin 1 beta, interleukin 2, interleukin 6, granulocyte-macrophage colony stimulating factor, tumor necrosis factor alpha, and interferon gamma), and soluble interleukin 2 receptors. Corticotropin, cortisol, and dehydroepiandrosterone sulfate (DHEAS) concentrations were also determined. Although the total number of lymphocytes decreased during fasting, NK cell activity increased significantly. Plasma cortisol and DHEAS concentrations also increased significantly whereas changes in corticotropin concentrations were not significant. The total number and percentage of CD4 cells decreased significantly during fasting but no other lymphocyte subsets changed significantly. The percentage of CD4 cells was negatively correlated with cortisol concentrations during fasting. No detectable changes occurred in cytokines or soluble interleukin 2 receptors during the study. All measured immunoendocrine values that changed during fasting returned to pre-fasting values during the re- feeding period. These findings indicate that fasting affects immune variables such as T cell subsets and NK cell activity at least in part through changes in adrenal gland-related hormones. Serum DHEAS increase from ~5.5 to ~8 μmol/L was associated with increased NK cell cytotoxicity from ~16 CU to ~28 CU, being a ratio of x 1.75. April 2009 Cytotoxic potential in mAb therapy : 26 Colin M Perrott
  • 27. A MORE EXACTING STUDY; S. B. Solerte et al, Dehydroepiandrosterone Sulfate Enhances Natural Killer Cell Cytotoxicity in Humans Via Locally Generated Immunoreactive Insulin-Like Growth Factor I, J Clin Endocrinol Metab 84: 3260–3267, 1999 Experimental and clinical investigations suggest the hypothesis that dehydroepiandrosterone sulfate (DHEAS) can positively influence natural killer (NK) immunity via locally produced insulin-like growth factor I (IGF-I) from NK cells. In the present study, the NK cell cytotoxicity (NKCC) and IGF-I levels in the supernatant of NK cells were studied at baseline and after exposure to various molar concentrations of DHEAS (from 1025-1028 mol/LzmL/7.75 3 106 NK cells) in healthy subjects of young and old age. DHEAS-induced NKCC was also determined after DHEAS coincubation with somatostatin-14 (1026 mol/LzmL/7.75 3 106 NK cells) and with interleukin-2 (IL-2; 100 IU/mLz7.75 3 106 NK cells). NK cells were previously isolated by Ficoll-Hypaque density gradient and then by immunomagnetic procedure; the purity obtained was 97 6 1%. NKCC was determined against K562 tumoral targets. We observed that the increase in NKCC after DHEAS exposure was dose dependent and was correlated with the amount of IGF-I released in the supernatant of cultured NK cells. NKCC and IGF-I generation from NK cells were more elevated in healthy elder subjects than in healthy young subjects. The coincubation of DHEAS with somatostatin-14 (SST) significantly suppressed NKCC and IGF-I release from NK in both groups, whereas higher NKCC was found after DHEAS plus IL-2 exposure than after incubation with DHEAS alone. Taken together, this study suggests a role for NK-generated IGF-I in the modulation of NKCC by DHEAS in humans. Although DHEAS may contribute to the IL-2-mediated NKCC, its activity on NK cytolytic function can be dependent on a autocrine mechanism (IGF-I-mediated), probably independent of cytokine activation. The higher NKCC response to DHEAS found in old subjects than in younger might counterbalance the age-dependent decline in circulating DHEAS, thus contributing to maintain the pattern of NK immunity during aging. Note: NK cells were separated from patient’s PMBC, suspended in a standard medium and then tested for cytotoxicity with the medium alone and also with additions of laboratory supplied DEAS, IL-2, and SST. Patient BMI was ~22 across the cohort. April 2009 Cytotoxic potential in mAb therapy : 27 Colin M Perrott