Anderson/Krummel
Ipilimumab Questions:
Topic 1 (Figures:1-2)
In this in vitro incubations, T cell responses are altered by varying CD28 and CTLA-4
engagement. Describe how this is working and draw a model. Conversely, in the in
vivo models (slide 2), T cell responses to a superantigen are also modified by these
exact same antibodies. How does this compare to the in vitro and draw a
mechanism. (superantigen, SEB, stimulates all T cells bearing the V8 gene
segment)
Review for the class the normal expression of CD28 and CTLA-4 during the arc of an
immune response. Describe which molecule is more likely critical for one phase
versus another. Draw this as a timeline.
Topic 2 (Figures:3-4)
In slide 3 mouse experiments, describe how antibodies are likely functioning in vivo,
based on the models of Slides 1-2. What other types of responses might be taking
place globally in these treated mice?
In slide 4 human experiments, why might only some people respond? Describe at
least three reasons. What kinds of immune monitoring might help you highlight and
recognize responders at 12 months compared to baseline?
Topic 3 (Figures:5-6)
In slide 5, in mouse experiments, tumor responses are shown for the same
antibodies containing different Fc tails. (IgG2a fixes complement). Why do some
antibodies work and some do not? Does this data fit the paradigms established in
Topic 1 and 2. What cell type(s) might be susceptible in this case and how does this
offer another perspective on anti-CTLA-4 antibodies.
In slide 6, another alternative mechanism for CTLA-4 function is provided. Observe
the cells in the figures and provide an interpretation of what you see. What would
be the significance for CTLA-4 mediated loss of CD86 expression for an immune
response.
Figure Legends for Slides:
Slide 1 (top): Influence of anti-CTLA-4 and anti-B7 antibodies on proliferative
responses of purified lymph node T cells. 105 BALB/c LN T cells were cultured for
72 h in 96-weU microwells coated with 0.1 g/ml anti-CD3. Anti-CD28 was used at a
1:1,000 dilution of ascites and B7 antibodies were used at 2.5 and 10 g/ml for antiB7-1 and anti-B7-2, respectively. Anti-CTLA-4 antibody 9H10 was added at 10
g/ml.
Slide 1 (bottom): Proliferation in response to a constant CD3 signal is affected by
the relative concentrations of CD28 and CTLA-4 signals. 107 5 microspheres
were coated with 1 g/ml of anti-CD3, the indicated concentrations of anti-CD28
and anti-CTLA-4, and control hamster Ig constituting a total antibody concentration
of 5 g/ml. 105 coated spheres were incubated in 96-well cultures with 105 purified
LN T calls for 72 h, pulsed with 1 Ci [3H]thymidine, and harvested after an
additional 16 h.
Slide 2: Kinetics of the expansion of VB8+ T cells in response to SEB and antibodies
to CD28 and CTLA-4 Age-matched BALB/c females were injected with 200 g of the
indicated antibodies followed by PBS or SEB. At the indicated time after injection,
spleens were harvested and analyzed for cellularity of the percentage of V8+ T cells.
Two mice were analyzed for each time point and data represents an average of the
values obtained. This experiment is representative of three independent
experiments
Slide 3: Treatment with anti-CTLA-4 reduces the growth of the murine
fibrosarcoma Sal N. Groups of five mice were injected subcutaneously in the flank
with a suspension of 1 x 106 Sal N fibrosarcoma cells. Treated groups were injected
intraperitoneally with 100 g of anti-CTLA-4 or irrelevant hamster control antibody
at days 0, 3, and 6 as indicated by the arrows. All control animals were killed by day
30. Two of five animals treated with anti-CTLA-4 remained tumor-free at day 55.
Slide 4: The median follow-up for overall survival (Panel A) in the ipilimumab (Ipi)plus-glycoprotein 100 (gp100) group was 21.0 months, and the median overall
survival was 10.0 months (95% CI, 8.5 to 11.5); in the ipilimumab-alone group, the
median follow-up was 27.8 months, and the median overall survival, 10.1 months
(95% CI, 8.0 to 13.8); and in the gp100-alone group, the median follow-up was 17.2
months, and the median overall survival, 6.4 months (95% CI, 5.5 to 8.7). The
median progression-free survival (Panel B- not shown on your PowerPoint) was
2.76 months (95% CI, 2.73 to 2.79) in the ipilimumab-plus-gp100 group, 2.86
months (95% CI, 2.76 to 3.02) in the ipilimumab-alone group, and 2.76 months
(95% CI, 2.73 to 2.83) in the gp100-alone group. The rates of progression-free
survival at week 12 were 49.1% (95% CI, 44.1 to 53.9) in the ipilimumab-plusgp100 group, 57.7% (95% CI, 48.9 to 65.5) in the ipilimumab-alone group, and
48.5% (95% CI, 39.6 to 56.7) in the gp100-alone group.
Slide 5: Isotype-dependent activity ofCTLA-4 antibodies in subcutaneous tumor
models. A, activity of anti-CTLA-4 isotypes inMC38 adenocarcinoma model in
C57BL/6 mice. B, activity of anti-CTLA-4 isotypes in CT26 adenocarcinoma model in
BALB/c mice. C57BL/6 or BALB/c mice were injected subcutaneously with 2 x 106
MC38 or 1 x 106 CT26 cells on day 0. On day 7, tumors were measured,
randomized, and then treated with the designated antibody (200 g/dose i.p.) and
again on days 10 and 14 for MC38 and days 10, 14, and 17 for CT26. Tumor volumes
were measured 3 times weekly. The number of tumor-free (TF) mice per group is
shown for each group. The experiment shown is representative of at least 3
different experiments using 10 mice per group. For CT26, tumor growth with antiCTLA-4-IgG1 and anti-CTLA-4-IgG1-D265A were equivalent (not shown).
Slide 6 (right): Confocal micrographs of adherent CD86-expressing CHO cells and
CTLA-4+ CHO cells after overnight incubation. CD86 (green) and CTLA-4 (red) were
detected through antibody staining. Colocalization of CD86 and CTLA-4 is shown in
yellow. (Bottom) An enlargement of the boxed area, with single-color images shown
in white for equal contrast. CHO-CD86 cultured alone are shown in fig. S7A.
Slide 6 (left): Flow cytometric analysis of CD86 surface expression on CHO-CD86
cells co-incubated with increasing numbers of untransfected (control), wild-type
CTLA-4, or CTLA-4 del36 cells (expressed as percent of CTLA-4+ cells in the
coculture). Surface CD86 was detected through antibody staining.
References for Figures
1. Krummel, M.F., and Allison, J.P. (1995). CD28 and CTLA-4 deliver opposing
signals which regulate the response of T cells to stimulation. Journal of
Experimental Medicine 182, 459-465.
2. Krummel, M.F., Sullivan, T.J., and Allison, J.P. (1996). Superantigen responses and
costimulation: CD28 and CTLA-4 have opposing effects on T cell expansion In Vitro
and In Vivo. IntImmunol 8, 101-105.
3. Leach, D.R., Krummel, M.F., and Allison, J.P. (1996). Enhancement of antitumor
immunity by CTLA-4 blockade. Science 271, 1734-1736.
4. Hodi, F.S., O'Day, S.J., McDermott, D.F., Weber, R.W., Sosman, J.A., Haanen, J.B.,
Gonzalez, R., Robert, C., Schadendorf, D., Hassel, J.C., et al. (2010). Improved Survival
with Ipilimumab in Patients with Metastatic Melanoma. New England Journal of
Medicine 363, 711-723.
5. Qureshi, O.S., Zheng, Y., Nakamura, K., Attridge, K., Manzotti, C., Schmidt, E.M.,
Baker, J., Jeffery, L.E., Kaur, S., Briggs, Z., et al. (2011). Trans-endocytosis of CD80 and
CD86: a molecular basis for the cell-extrinsic function of CTLA-4. Science 332, 600603.
6. Selby, M.J., Engelhardt, J.J., Quigley, M., Henning, K.A., Chen, T., Srinivasan, M., and
Korman, A.J. (2013). Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor
activity through reduction of intratumoral regulatory T cells. Cancer immunology
research 1, 32-42.