Immunogenicity of KH1KLH Conjugate plus QS21 Adjuvant Vaccine in Patients with Ovarian Cancer - PowerPoint PPT Presentation

Title: Immunogenicity of KH1KLH Conjugate plus QS21 Adjuvant Vaccine in Patients with Ovarian Cancer


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Immunogenicity of KH-1-KLH Conjugate plus QS-21
Adjuvant Vaccine in Patients with Ovarian Cancer
Lauren Andrews1, Sara Blum1, Chandra Hood1,
Govind Ragupathi1, Maria Spassova2, Prashant P.
Deshpande3, Samuel J. Danishefsky3 and Philip O.
Livingston1
Laboratories of 1Tumor Vaccinology, 2Synthetic
Core Facility and 3Bioorganic Chemistry, Memorial
Sloan-Kettering Cancer Center, 1275 York Avenue,
New York, New York 10021
Analysis Methods Serological Assays
Enzyme Linked Immunosorbent Assay
Fluorescence Activated Cell Scanning Assay
A KH-1KLH conjugate vaccine with immunological
adjuvant QS-21 was tested in 8 patients with
ovarian cancer in first remission. KH-1 is an
analog of the Ley glycolipid antigen
over-expressed on the surface of ovarian, breast,
prostate, and other epithelial cancer cells.
This study represents an attempt to improve upon
the immunogenicity observed in the previous study
of the LeyKLH vaccine to determine which
construct should be included in a subsequent
polyvalent vaccine clinical trial. A previous
murine study showed that the KH-1KLH conjugate
induces antibodies recognizing not only KH-1
antigen but also Ley antigen. In mice, KH-1KLH
results in much higher anti-Ley IgM and IgG
antibody titer levels than those observed in
response to immunization with the LeyKLH
construct. The present phase I pilot study tests
whether KH-1KLH can similarly overcome the low
immunogenicity of Ley in patients. Patients were
immunized subcutaneously on weeks 0, 1, 2, 6, and
14 with KH-1 (30 µg) in KH-1KLH conjugate plus
saponin adjuvant QS-21 (100 µg). Sera were tested
by ELISA against Leyceramide and KLH starting at
a 120 serial dilution, and cell surface
reactivity was determined by FACS with OVCAR-3
(ovarian cancer) cells. ELISA results show that
KH-1KLH induced very low IgM titers and
undetectable IgG titers in all patients (anti-Ley
median pre/post peak titers IgG 0/0, IgM
0/20). FACS at a 120 sera dilution showed that
these antibodies were slightly reactive with Ley
positive OVCAR-3 cells. A large anti-carrier
molecule response to the KH-1KLH vaccine was
shown by ELISA assays with KLH antigen, anti-KLH
IgM and IgG median peak titers of 12560. No KH-1
antigen was available to test whether anti-KH-1
antibodies were produced. However, KH-1KLH was
found to have a lower Ley immunogenicity than
LeyKLH in patients. Therefore, KH-1KLH will not
be included in the upcoming clinical trial of our
polyvalent ovarian cancer vaccine.
live cancer cell expressing target surface
antigen (Ley) OVCAR-3 (ovarian cancer) and
MCF-7 (breast cancer)
Plate Reader Measures the Intensity of Yellow
Color (Absorbance at ? 405 nm)
Ley-ceramide or KLH
Target Cell
Target Antigen
Human serum albumin (HSA)
Antibody for target antigen (a-Ley) Testing for
presence in sera sample!
Blocking Molecule
1o Antibody
Antibody that recognizes Fc region of human
immunoglobulin with fluorescent tag FITC Goat
a-Human IgG or IgM
Flow Cytometer Measures Fluorescence of a Single
Stream of Cells
Antibody that recognizes Fc region of 1o
antibody with linked enzyme Goat anti-Human IgG
or IgM
2o Antibody
52.1 Cells Positive
2o Antibody
Phosphatase substrate dissolved in
diethanolamine Catalyzes reaction producing
yellow color absorbed at 405nm
Chromogenic Substrate
Single Well of 96-Well Plate After carrying out
ELISA Assay
Pre Sera 10 Post Sera
Results
Background
Conjugate Molecule
Surface Cancer Antigen Vaccine
Cell Surface Antigens
Anti-antigen Immunoglobulin

• Carrier Protein Keyhole Limpet Hemocyanin
  (KLH)
• KLH is a mollusk protein
• Large, foreign protein easily recognized by
  immune system
• Carrier protein purpose Amplify Immune
  Response

Vaccine Induces Antibody Production
Ovarian Cancer Tumor Cells

• Immunological Adjuvant QS-21
• QS-21 is a saponin extracted from bark the of a
  
• Quillaja South American soap tree
• Adjuvant purpose Amplify Immune Response

Table 1 Proportion of Tumor Specimens with 50
or More of Cells Positive by Immunohistology
Antigen (mAb)
All the tumor tissues were stained by
Avidin-Biotin Complex immunoperoxidase methods.
Results Summary
Lewis-y expressed in about 80 of ovarian cancer
tumors in over 50 of the cells.

• 4 out of 8 patients had no Ley immune response,
  as detected by ELISA.
• Peak KLH titers are on the order of 1000 to
  10,000 times greater than peak Ley titer.
• Anti-KLH antibody titers do not directly
  correlate to trends in anti-Ley antibody titers
  for the same patient.
• The vaccine did not induce measurable anti-Ley
  IgG antibody production in any of the patients.
• Ley immune response is short-lived, whereas KLH
  response is sustained at much higher levels for a
  longer duration.
• Sera samples were slightly reactive with
  OVACAR-3 cell surface, and negligibly reactive
  with MCF-7 cells.

Zhang S, Zhang HS, Cordon-Cardo C, Reuter VE,
Singhal AK, Llyod KO, Livingston PO. Selection of
tumor antigens as targets for immune attack using
immunohistochemistry II .blood group-related
antigens. Int J Cancer 73 50-56, 1997.
KH-1-KLH Vaccine
KH-1 Molecule
Lewis-y (Ley) Molecule
KH-1 Lewis-y Lewis-x Analog

• In a previous clinical trial, the Ley-KLH
  vaccine was found to have a low immunogenicity in
  patients.
• This study of KH-1-KLH is an attempt to induce
  higher levels of anti-Ley immunoglobulin.
• The previous mouse experiment with the KH-1-KLH
  vaccine showed that
• 1. KH-1 antibodies
  recognized both KH-1 and Ley.
• 2. KH-1-KLH induces
  higher anti-Ley antibody titers than the Ley-KLH
  vaccine.
• Thus, the KH-1-KLH vaccine proceeded to the phase
  I clinical trial presented here.

Conclusions
Spassova MK, Bornmann WG, Ragupathi G, Sukenick
G, Livingston PO, and Danishefsky SJ. Synthesis
of selected Ley and KH-1 analogues a medicinal
chemistry approach to vaccine optimization. J Org
Chem 70 3383-3395, 2005.
Vaccine Formula 1 Dose

100µg QS-21
Acknowledgements Gerstner Sloan-Kettering
Graduate School of Biomedical Sciences Summer
Undergraduate Research Program '05 and the whole
Livingston Lab crew!