Therapy Of Cancer: Which Cells Are Important, And How To Assay Their Activity
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1 ADCC Effector Cells In Antibody Therapy Of Cancer: Which Cells Are Important, And How To Assay Their Activity M. Jules Mattes
2 Potential ADCC effector cells and their Fcγ receptors Cell type Fcγ receptors NK cells FcγRIIIA only, an activating receptor Neutrophils Four FcγR, including the inhibitoryreceptor receptor FcγIIB Macrophages Five FcγR, including the inhibitory receptor FcγIIB
3 1. Almost all in vitro assays for ADCC, using human blood leukocytes, detect only NK cells (even though neutrophils and monocytes are present) 2. There is strong evidence that the primary effector cells in vivo in Ab-mediated tumor therapy are mϕs, not NK cells
4 Why are the other effector cell types not assayed?
5 Evidence from TF Tedder, using Abs to mouse CD20, that mϕ are important in ADCC, while NK cells are not: 1. Therapy normal in mice with deficient NK cells (beige or perforin mutated) 2. Therapy inhibited by clodronate- encapsulated liposomes 3. Therapy enhanced by using mice lacking the inhibitory FcγRIIB
6 Evidence from JV Ravetch that mϕ are important t in ADCC, while NK cells are not: 1. Therapy enhanced by using mice lacking the inhibitory FcγRIIB 2 Th li i i hi hth i 2. Therapy normal in mice in which there is no Fcγ receptor on NK cells
7 Deficiency of experimental models that have been used to determining the mechanism of action of Abs 1. Isolated tumor cells vs. established tumors 2. Sublethal irradiation sometimes required to allow tumor growth
8 Therapy of measurable sc RL in scids with 2.0 unconjugated Abs % Su urviving mic ce w/o tumo ors >1.0 cm control 60 L243 L243-gamma4 modified ha20 anti CD20 anti CD74 hll P< Days after Ab injection
9 Why is human IgG4 so effective? 1. Intended to have fewer side effects, so should be less effective. 2 Hypothesis: binds to activating FcγR but 2. Hypothesis: binds to activating FcγR but not to inhibitory FcγR, due to species differences.
10 Therapy of measurable RL sc in common gamma KO/rag mice with 1.0 mg anti-cd20 Ab 2.0 control 2.0 anti-cd20 Tumor vo olume (cm3) Days after Ab injection Mice: Taconic Emerging Model Double Knockout Mice Common gamma (γc)/rag2 (This γ chain is a subunit of cytokine receptors, not Fc receptors)
11 Why is therapy so effective in mice lacking NK cells? 1. NK cells inhibit therapy? 2. Macrophages or other effector cells are spontaneously sl activated ated in these mice.
12 Wh t NK ll ff ti Why aren t NK cells effective in tumor therapy?
13 Evidence that NK cells or neutrophils are responsible for Ab therapy: 1. Removal of these specific cells by injecting Abs that react with them inhibits Ab therapy Hernandez-Ilizaliturri et al, Clin Cancer Res 9: 5866, 2003 Cittera et al, J Immunol 178: 6616, 2007
14 Possible side- effects of depleting effector cells with Abs 1. Blocking FcR non-specifically 2. Activating and depleting complement
15 Hypothesis: Abs to mouse RBC (such as TER-119) would inhibit Ab therapy by blocking FcR or depleting complement
16 Conclusions 1. Further studies are required to definitively identify the key effector cells in ADCC in vivo. 2. These effector cells should then be used as effector cells in in vitro assays for potency.
17 Possible sources of mϕ effector cells 1. Human: peripheral blood monocytes, but these must be cultured in vitro 2. Mouse, rat or other animal: peritoneal exudate cells or spleen cells 3. Can animal cells be used as a surrogate for human?
18 Assays for mϕ ADCC 1. Target cells are usually phagocytized, so typical assays detecting release of substances from target t cells will not work. 2. Assay growth of target cells. 3. Target cells can be labeled in various ways, to assess phagocytosis. But phagocytosis should be distinguished from binding.
19 Possible strategies for improved Ab therapy 1. Modify Abs to enhance their mechanisms of action 2. Stimulate the effector cells in vivo A. More cells B. Cells with greater activity 3. Enhance recruitment of effector cells into the tumor. Use complement?
20
21 Y Y
22 Is functional affinity valid? 1. Affinity must be equal to the ratio of the rate constants. t 2. The Scatchard plot must be linear. 3. Affinity must be independent of trivial experimental conditions, such as the volume used.
23 Conclusions Ab dissociation from the cell surface is very slow, essentially irreversible. ibl Functional affinities are invalid. Parameters of Fab binding do not apply to the binding of the original intact IgG.
24 Which binding parameters should be measured? Dissociation of bound Ab over 2-3 days or more, until most of the bound Ab has dissociated (not a rate constant) Use natural tumor cells as targets. Respectable Abs bind essentially irreversibly
25
26 Are Fc functions required? 1. The example of panitumumab, an IgG2 Ab to EGFr. IgG2 Abs do not activate any of the Fc functions. 2. Effect of Fcγ genetic polymorphisms on therapy. 3 Does this depend on the particular 3. Does this depend on the particular tumor?
27 In depleting CD20 B cells, both type I and type 2 anti-cd20 Abs operate exclusively via activatory Fcγ receptor-expressing macrophages. SA Beers,..MJ Glennie, MS Cragg, Blood, 115: 5191, 2010
28 Obstacles to Ab therapy of cancer that do not apply to a 1-day old tumor 1. Ab penetration of the tumor mass 2. Presence of effector cells and/or proteins, such as complement proteins, at the site of Ab binding within the tumor
29 Will studies with NK cell ADCC provide useful information regarding mϕ ADCC? 1. They will confirm that the Fc region, as well as the rest of the Ab, is generally intact. 2. Because of the different Fc receptors, and the different cell physiology, they cannot provide reliable data on function.
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